Methods for identifying compounds for treatment of cell proliferative disorders associated with adaptor protein interactions

ABSTRACT

The present invention relates to compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or protein tyrosine phosphatase capable of complexing with a member of the SH2- and/or SH3-containing family of adaptor proteins is involved. This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL tyrosine kinase product in vivo and is necessary for the activation of the oncogenic potential of the BCR/ABL product. The present invention further relates to protein tyrosine kinase/adaptor protein complexes and the uses of these complexes for the identification of agents capable of decreasing or inhibiting the interaction between the members of such complexes.

This is a division of application Ser. No. 08/449,648, filed May 24,1995 now abandoned which is a division of application Ser. No.08/246,441 filed May 20, 1994 now U.S. Pat. No. 6,066,463, which is acontinuation-in-part of Ser. No. 08/127,922 filed Sep. 28, 1993, nowabandoned, each of which is incorporated herein in their entirety.

1. INTRODUCTION

The present invention relates to compositions and methods for theprevention and treatment of cell proliferative disorders wherein aprotein tyrosine kinase or protein tyrosine phosphatase capable ofcomplexing with a member of the SH2- and/or SH3-containing family ofadaptor proteins is involved. This invention is based, in part, on thesurprising discovery that the adaptor protein, GRB-2, binds theintracellular BCR-ABL tyrosine kinase product in vivo and is necessaryfor the activation of the oncogenic potential of the BCR-ABL product andthat disruption of the signaling capability of GRB-2 can reverse thetransformed phenotype of cells and reduce tumor growth in animals. Thepresent invention further relates to protein tyrosine kinase/adaptorprotein complexes and the uses of these complexes for the identificationof agents capable of disrupting the interaction between the members ofsuch complexes.

2. BACKGROUND

2.1. Protein Phosphorylation and Signal Transduction

Cells rely, to a great extent, on extracellular molecules as a means bywhich to receive stimuli from their immediate environment. Theseextracellular signals are essential for the correct-regulation of suchdiverse cellular processes as differentiation, contractility, secretion,cell division, contact inhibition, and metabolism. The extracellularmolecules, which can include, for example, hormones, growth factors,lymphokines, or neurotransmitters, act as ligands that bind specificcell surface receptors. The binding of these ligands to their receptorstriggers a cascade of reactions that brings about both the amplificationof the original stimulus and the coordinate regulation of the separatecellular processes mentioned above. In addition to normal cellularprocesses, receptors and their extracellular ligands may be involved inabnormal or potentially deleterious processes such as virus-receptorinteraction, inflammation, and cellular transformation to a cancerousstate.

A central feature of this process, referred to as signal transduction(for recent reviews, see Posada et al., 1992, Mol. Biol. Cell 3:583-592;Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is thereversible phosphorylation of certain proteins.

The phosphorylation or dephosphorylation of amino acid residues triggersconformational changes in regulated proteins that alter their biologicalproperties. Proteins are phosphorylated by protein kinases and aredephosphorylated by protein phosphatases. Protein kinases andphosphatases are classified according to the amino acid residues theyact on, with one class being serine-threonine kinases and phosphatases(reviewed in Scott et al., 1992, 2:289-295), which act on serine andthreonine residues, and the other class being the tyrosine kinases andphosphatases (reviewed in Fischer et al., 1991, Science 253:401-406;Schlessinger et al., 1992, Neuron 9:383-391; Ullrich et al., 1990, Cell61:203-212), which act on tyrosine residues. Phosphorylation is adynamic process involving competing phosphorylation anddephosphorylation reactions, and the level of phosphorylation at anygiven instant reflects the relative. activities, at that instant, of theprotein kinases and phosphatases that catalyze these reactions.

While the majority of protein phosphorylation occurs at serine andthreonine amino acid residues, phosphorylation at tyrosine residues alsooccurs, and has begun to attract a great deal of interest since thediscovery that many oncogene products and growth factor receptorspossess intrinsic protein tyrosine kinase activity. The importance ofprotein tyrosine phosphorylation in growth factor signal transduction,cell cycle progression and neoplastic transformation is now wellestablished (Cantley et al., 1991, Cell 64:281-302; Hunter T., 1991,Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger et al.,1992, Neuron 9:383-391; Ullrich et al., 1990, Cell 61:203-212).Subversion of normal growth control pathways leading to oncogenesis hasbeen shown to be caused by activation or overexpression of proteintyrosine kinases which constitute a large group of dominant oncogenicproteins (reviewed in Hunter, T., 1991, Cell 64:249-270).

2.2. Protein Tyrosine Kinases

Protein tyrosine kinases comprise a large family of proteins, includingmany growth factor receptors and potential oncogenes, which shareancestry with, but nonetheless differ from, serine/threonine-specificprotein kinases (Hanks et al., 1988, Science 241:42-52). The proteinkinases may further be defined as being receptors or non-receptors.

Receptor-type protein tyrosine kinases, which have a transmembranetopology have been studied extensively. The binding of a specific ligandto the extracellular domain of a receptor protein tyrosine kinase isthought to induce receptor dimerization and phosphorylation of their owntyrosine residues. Individual phosphotyrosine residues of thecytoplasmic domains of receptors may serve as specific binding sitesthat interact with a host of cytoplasmic signalling molecules, therebyactivating various signal transduction pathways (Ullrich et al., 1990,Cell 61:203-212).

The intracellular, cytoplasmic, non-receptor protein tyrosine kinasesmay be broadly defined as those protein tyrosine kinases which do notcontain a hydrophobic, transmembrane domain. Within this broadclassification, one can divide the known cytoplasmic protein tyrosinekinases into four distinct morphotypes: the SRC family (Martinez et al.,1987, Science 237:411-414; Sukegawa et al., 1987, Mol. Cell. Biol.7:41-47; Yamanishi et al., 1987, 7:237-243; Marth et al., 1985, Cell43:393-404; Dymecki et al., 1990, Science 247:332-336), the FES family(Ruebroek et al., 1985, EMBO J. 4:2897-2903; Hao et al., 1989, Mol.Cell. Biol. 9:1587-1593), the ABL family (Shtivelman et al., 1986, Cell47:277-284; Kruh et al., 1986, Science 234:1545-1548), and the JAKfamily. While distinct in their overall molecular structure, each of themembers of these morphotypic families of cytoplasmic protein tyrosinekinases share non-catalytic domains in addition to sharing theircatalytic kinase domains. Such non-catalytic domains are the SH2 (SRChomology domain 2; Sadowski et al., Mol. Cell. Biol. 6: 4396-4408; Kochet al., 1991, Science 252:668-674) domains and SH3 domains (Mayer etal., 1988, Nature 332:269-272). Non-catalytic domains are thought to beimportant in the regulation of protein-protein interactions duringsignal transduction (Pawson et al., 1992, Cell 71:359-362).

While the metabolic roles of cytoplasmic protein tyrosine kinases areless well understood than that of the receptor-type protein tyrosinekinases, significant progress has been made in elucidating some of theprocesses in which this class of molecules is involved. For example, lckand fyn, members of the src family, have been shown to interact withCD4/CD8 and the T cell receptor complex, and are thus implicated in Tcell activation, (Veillette et al., 1992, TIG 8:61-66). Certaincytoplasmic protein tyrosine kinases have been linked to certain phasesof the cell cycle (Morgan et al., 1989, Cell 57:775-786; Kipreos et al.,1990, Science 248: 217-220; Weaver et al., 1991, Mol. Cell. Biol.11:4415-4422). Cytoplasmic protein tyrosine kinases have been implicatedin neuronal development (Maness, P., 1992, Dev. Neurosci. 14:257-270).Deregulation of kinase activity through mutation or overexpression is awell-established mechanism underlying cell transformation (Hunter etal., 1985, supra; Ullrich et al., supra).

2.3. G-Proteins and Signal Transduction

Guanine-nucleotide-binding proteins, (G-proteins; Simon et al., 1991,Science 252:802-808; Kaziro et al., 1991, Ann. Rev. Biochem. 60:349-400)such as Ras (for review, see Lowy et al., 1993, Ann Rev. Biochem.62:851-891), play an essential role in the transmission of mitogenicsignals from receptor tyrosine kinases. Taking Ras as an example, theactivation of receptor tyrosine kinases by ligand binding results in theaccumulation of the active GTP bound form of the Ras molecule (Gibbs etal., 1990, J. Biol. Chem. 265:20437-2044; Satoh et al., 1990, Proc.Natl. Acad. Sci. USA 87:5993-5997; Li et al., 1992, Science256:1456-1459; Buday et al., 1993, Mol. Cell. Biol. 13:1903-1910; Medemaet al., 1993, Mol. Cell. Biol. 13:155-162). Ras activation is alsorequired for transformation by viral oncogenic tyrosine kinases (Smithet al., 1986, Nature 320:540-43).

Ras activity is regulated by the opposing actions of theGTPase-activating proteins (GAPs) and guanine nucleotide exchangefactors, with GAPs stimulating the slow intrinsic rate of GTP hydrolysison Ras and exchange factors stimulating the basal rate of exchange ofGDP for GTP on Ras. Thus, GAPS act as negative regulators of Rasfunction, while exchange factors act as Ras activators.

Recently, a direct link between activated receptor tyrosine kinases andRas was established with the finding that the mammalian GRB-2 protein, a26 kilodalton protein comprised of a single SH2 and two SH3 domains(Lowenstein et al., 1992, Cell 70:431-442), directly couples receptortyrosine kinases to the Ras exchange factor Sos in mammals andDrosophila (Buday et al., 1993, Cell 73:611-620; Egan et al., 1993,Nature 363:45-51; Li et al., 1993, Nature 363:85-87; Gale et al., 1993,Nature 363:88-92; Rozakis-Adcock et al., 1993, Nature 363:83-85; Chardinet al., 1993, Science 260:1338-1343; Oliver et al., Cell 73:179-191;Simon et al., 1993, Cell 73:169-177). The GRB-2 SH2 domain binds tospecific tyrosine phosphorylated sequences in receptor tyrosine kinaseswhile the GRB-2 SH3 domains bind to proline-rich sequences present inthe Sos exchange factor. Binding of GRB-2 to the receptor kinases,therefore, allows for the recruitment of Sos to the plasma membrane,where Ras is located (Schlessinger, J., 1993, TIBS 8:273-275).

2.4. BCR-ABL in the Development of Leukemias

Activation of the oncogenic potential of normal cellular proteins suchas protein tyrosine kinases may occur by alteration of the proteins'corresponding enzymatic activities, their inappropriate binding to othercellular components, such as those mentioned above in Section 2.3, orboth.

For example, the BCR-ABL protein tyrosine kinase oncoprotein maytransform cells via changes in enzyme activity and/or altering ofnoncovalent protein-protein interactions. The gene encoding the BCR-ABLoncoprotein is a chimeric oncogene generated by the translocation ofsequences from the cABL protein tyrosine kinase on chromosome 9 into BCRsequences on chromosome 22 (reviewed in Kurzock et al., 1988, N. Engl.J. Med. 319:990-998, and Rosenberg et al., 1988, Adv. in Virus Res.35:39-81). The BCR-ABL oncogene has been implicated in thepathenogenesis of Philadelphia chromosome (Ph¹) positive humanleukemias. Namely, the Ph¹ chromosome is found in at least 90 to 95percent of cases of chronic myelogenous leukemia (CML), which is aclonal cancer arising from the neoplastic transformation ofhematopoietic stem cells (Fialkow et al., 1977, Am. J. Med. 63:125-130),and is also observed in approximately 20 percent of adults with acutelymphocytic leukemia (ALL), 5 percent of children with ALL, and 2percent of adults with acute myelogenous leukemia (AML) (Whang-Peng etal., 1970, Blood 36:448-457; Look, A. T., 1985, Semin. Oncol.12:92-104). The BCR-ABL gene produces two alternative chimeric proteins,P210 BCR-ABL, and P185 BCR-ABL, which are characteristic of CML and ALL,respectively. Further, it has recently been directly demonstrated thatthe BCR-ABL gene product is the causative agent in CML (Skorski et al.,1993, J. Clin Invest. 92:194-202; Snyder et al., 1993, Blood82:600-605).

Clinically, CML is characterized by a biphasic course. The diseasebegins with a chronic phase marked by a greatly increased pool ofuncommitted myeloid progenitor cells. Because terminal differentiationis maintained, this results in greatly increased pools of circulatingmature granulocytes. After a period of several weeks to many years, astate of accelerated myeloproliferation develops wherein the myeloidcells progressively lose their capacity for terminal differentiation.During this time, thrombocytosis, basophilia, and clonal cytogeneticabnormalities often appear. These abnormalities signal the terminal,blast-crisis stage, during which immature blast cells rapidlyproliferate and the patient inevitably dies.

It has previously been shown that the BCR-ABL proteins exhibitheightened tyrosine kinase and transforming capabilities compared to thenormal c-ABL protein (Konopka et al., 1984, Cell 37:1035-1042). BCRfirst exon sequences specifically activate the tyrosine kinase andtransforming potential of BCR-ABL (Muller et al., 1991, Mol. Cell. Biol.11:1785-1792; McWhirter et al., 1991, Mol. Cell. Biol. 11:1553-1565).The BCR first exon is capable of binding to the ABL SH2 domain in aphosphotyrosine-independent manner (Pendergast et al., 1991, Cell66:161-171), and deletion of BCR sequences essential for ABL SH2-bindingrender BCR-ABL nontransforming (Pendergast et al., 1991, Cell66:161-171). In addition, it has been demonstrated that BCR binds, invitro, to some other SH2 domains encoded by other proteins (Muller etal., 1992, Mol. Cell. Biol. 12:5087-5093). While one may infer fromthese results that some aspect of SH2 domain-binding to BCR is involvedin the oncogenicity of the BCR-ABL oncoprotein, the mechanism by whichsuch BCR-ABL oncogenesis occurs is still obscure. For example, given themyriad of SH2-containing proteins which are known to exist, theidentification of a BCR-ABL effector(s) will necessitate much furtherstudy.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of cell proliferative disorders wherein aprotein tyrosine kinase or a protein tyrosine phosphatase capable ofcomplexing with a member of the SH2- and/or SH3-containing family ofadaptor proteins is involved. The present invention further relates toprotein tyrosine kinase/adaptor protein complexes, protein tyrosinephosphatase/adaptor protein complexes, and the uses of these complexesfor the identification of agents capable of disrupting the interactionbetween the components of such complexes.

“Protein tyrosine kinase” will, herein, be abbreviated “PTK”, and“protein tyrosine phosphatase” will, herein be abbreviated “PTP”. It isto be understood that “PTK” may refer to either a transmembrane,receptor-type protein tyrosine kinase or a cytoplasmic protein tyrosinekinase, unless otherwise indicated, and, likewise, “PTP” may refer toeither a transmembrane, receptor-type protein tyrosine phosphatase or acytoplasmic protein tyrosine phosphatase, unless otherwise indicated.

This invention is based, in part, on the surprising discovery that theadaptor protein, GRB-2, binds the intracellular BCR-ABL PTK product invivo and is necessary for the activation of the oncogenic potential ofthe BCR-ABL product. The inventors are the first to demonstrate thephysiological relevance of the interactions between the members of asignal transduction pathway, in part by showing that disruption of thissignal transduction can result in the reversal of the transformedphenotype of cells and inhibit tumor growth in animals. The datarepresenting this discovery is presented in the Working Examples inSections 6 through 12, below. The invention, therefore, represents thefirst instance whereby the SH2- and/or SH3-containing adaptor family ofproteins, especially the GRB-2 member of the GRB subfamily of proteins,are implicated in the development and maintenance of cellproliferation/activation—herein demonstrated for the abnormal cellularproliferation involved in oncogenesis, the transformation process, andthe development of human cancer. Still further, with respect to BCR-ABLtransformation, the present invention discloses the first effector(i.e., GRB-2) for the BCR-ABL product.

3.1. Abbreviations

The following table lists the single-letter and triple-letterabbreviations for amino acids that are in common use among proteinchemists and that are used herein.

Amino Acid One Letter Code Three Letter Code Alanine A Ala Arginine RArg Asparagine N Asn Aspartic Acid D Asp Cysteine C Cys Glutamic Acid EGlu Glutamine Q Gln Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methioine M Met Phenylalanine F Phe Proline PPro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr ValineV Val Not Specified X

4. DESCRIPTION OF THE FIGURES

FIG. 1. Conserved motifs of the catalytic domains of PTKs (SEQ ID NOS:11-17). (From Wilks, A. F., 1990, Progress in Growth Fact. Res.2:97-111)

FIG. 2. BCR-ABL binds to the GRB-2 adaptor protein in living cells.K562, MEG-01 and ALL-1 Ph¹ hematopoietic cells and Ratl fibroblastsexpressing P185 BCR-ABL, P210 BCR-ABL, and P160 v-abl proteins werelysed and the lysates incubated with pre-immune sera (lanes 1, 4, 7, 10,13, 16), anti-ABL pEX4 antibodies (lanes 2, 5, 6, 11, 14, 17) andanti-GRB2 antibodies (lanes 3, 6, 9, 12, 15, 18). The immunoprecipitateswere collected on protein A-Sepharose beads and subjected to in vitrophosphorylation in the presence of [γ³²-P] ATP and MnCl₂. The reactionswere terminated after 30 min. at 30° C., washed and analyzed by SDS 8%polyacrylamide gel electrophoresis. The ³²P-labeled proteins weredetected by autoradiography.

FIG. 3. GRB-2 co-immunoprecipitates with BCR-ABL. MEG-01 cells werelysed and the lysate incubated with pre-immune sera (lane 1), anti-ABLpEX4 antibodies (lane 2) and anti-GRB-2 antibodies (lane 3). Theimmunoprecipitated proteins were separated by SDS 12.5% polyacrylamidegel electrophoresis, transferred to nitrocellulose and immunoblottedwith anti-GRB-2 antibodies. Bound antibodies were visualized with [¹²⁵I]protein A.

FIG. 4. BCR-ABL binds to GRB-2 in vitro. [³⁵S] methionine-labeledproteins from lysates of Sf9 insect cells infected with P185 BCR-ABLrecombinant baculovirus were incubated with equal amounts of immobilizedGST alone (lane 1), GST-GRB-2 full length (lane 2), GST-amino-terminal(N) GRB-2 SH3 domain (lane 3), GST-GRB-2 SH2 domain (lane 4),GST-carboxy-terminal (C) GRB-2 SH3 domain (lane 5) and anti-ABLantibodies bound to protein A-Sepharose beads (lane 6). After incubationfor 90 min. at 4° C., the beads were washed four times with incubationbuffer and twice with RIPA buffer to remove unbound material. Boundproteins were analyzed by SDS 7% polyacrylamide gel electrophoresis anddetected by fluorography.

FIG. 5A. SH2 domain-mediated binding of GRB-2 to BCR-ABL requirestyrosine phosphorylation of BCR sequences. Sf9 insect cells were singlyinfected with cABL recombinant baculoviruses. Three days post-infection,the cells were labeled with [³⁵S] methionine. The labeled cells werelysed and the lysate proteins incubated with GST alone (lanes 1),GST-GRB-2 full length (lanes 2), GST-GRB-2 N SH3 (lanes 3), GST-GRB-2SH2 (lanes 4), GST-GRB-2 C SH3 (lanes 5) and the corresponding anti-ABL(lane 6) antibodies in the presence of protein A-Sepharose beads. Afterincubation for 90 min. at 4° C., the beads were washed four times withincubation buffer and twice with RIPA buffer. Bound proteins wereanalyzed by SDS 10% polyacrylamide gel electrophoresis and detected byfluorography.

FIG. 5B. SH2 domain-mediated binding of GRB-2 to BCR-ABL requirestyrosine phosphorylation of BCR sequences. Sf9 insect cells were singlyinfected with CBCR recombinant baculoviruses. Three days post-infection,the cells were labeled with [³⁵S] methionine. The labeled cells werelysed and the lysate proteins incubated with GST alone (lanes 1),GST-GRB-2 full length (lanes 2), GST-GRB-2 N SH3 (lanes 3), GST-GRB-2SH2 (lanes 4), GST-GRB-2 C SH3 (lanes 5) and anti-BCR (lane 6)antibodies in the presence of protein A-Sepharose beads. Afterincubation for 90 min. at 4° C., the beads were washed four times withincubation buffer and twice with RIPA buffer. Bound proteins wereanalyzed by SDS 10% polyacrylamide gel electrophoresis and detected byfluorography.

FIG. 5C. SH2 domain-mediated binding of GRB-2 to BCR-ABL requirestyrosine phosphorylation of BCR sequences. Sf9 insect cells werecoinfected with cABL and cBCR recombinant baculoviruses. Three dayspost-infection, the cells were labeled with [³⁵S] methionine. Thelabeled cells were lysed and the lysate proteins incubated with GSTalone (lanes 1), GST-GRB-2 full length (lanes 2), GST-GRB-2 N SH3 (lanes3), GST-GRB-2 SH2 (lanes 4), GST-GRB-2 C SH3 (lanes 5) and thecorresponding anti-ABL and anti-BCR (lane 6) antibodies in the presenceof protein A-Sepharose beads. After incubation for 90 min. at 4° C., thebeads were washed four times with incubation buffer and twice with RIPAbuffer. Bound proteins were analyzed by SDS 10% polyacrylamide gelelectrophoresis and detected by fluorography.

FIG. 6A. GRB-2 forms a complex with the chimeric BCR-ABL tyrosine kinasebut not with cABL and CBCR sequences in vivo. COS cells were transfectedwith P185 wild type (lane 1-3) and cABL (lanes 4-6) cDNAs cloned intothe pSRα vector. Three days post-transfection, the cells were lysed andthe lysates incubated for 2 hrs. at 4° C. with normal rabbit sera (NRS)(lanes 1 and 4), anti-ABL 2/3 antibodies (lanes 2 and 5) and anti-GRB-2antibodies (lanes 3 and 6). The immunoprecipitates were collected onprotein A-Sepharose beads and washed six times with incubation buffer.Bound proteins were analyzed by SDS 8% polyacrylamide gelelectrophoresis, transferred to nitrocellulose filters and the filtersincubated with anti-ABL mouse monoclonal antibody. Immunoreactive bandswere visualized with the enhanced chemiluminescence (ECL) detectionsystem (Amersham).

FIG. 6B. GRB-2 forms a complex with the chimeric BCR-ABL tyrosine kinasebut not with cABL and cBCR sequences in vivo. COS cells were transfectedwith BCR (Δ872-1271) cDNA cloned into the pSRα vector. The cells werelysed three days post-infection and the lysates incubated with controlsera (NRS) (lane 1), anti-BCR antibodies (lanes 2), or anti-GRB-2antibodies (lane 3) for 2 hrs. at 4° C. The immunoprecipitates werecollected with protein A-Sepharose beads, washed four times withincubation buffer and twice with 50 mM Tris-HCl, pH 7.0 and thensubjected to in vitro phosphorylation in the presence of γ-³²P-ATP andMnCl₂. Proteins were analyzed by SDS 8% polyacrylamide gelelectrophoresis and autoradiography.

FIG. 7. Phosphopeptide maps of wild type and (Y177F) forms of BCR-ABL.COS cells were transfected with P185 wild type and P185 (Y177F) clonedinto the pSRαMSVtkneo vector. Three days post-transfection, the cellswere lysed and the lysates incubated with anti-ABL antibodies. Theimmunoprecipitates were subjected to in vitro autophosphorylation in thepresence of [γ-³²]ATP and MnCl₂. Proteins were analyzed bySDS-polyacrylamide gel electrophoresis, transferred to nitrocelluloseand subjected to phosphopeptide mapping. A single phosphopeptide whichis present in the wild type P185 protein, but is absent from the mutantP185 (Y177F) protein is indicated by arrows. The experiment shown wasbeen exposed to autoradiography for 16 hr.

FIG. 8A. Mutation of tyrosine 177 to phenylalanine in the BCR first exonabolishes binding of the GRB-2 SH2 domain to BCR-ABL. Sf9 insect cellswere co-transfected with wild type baculovirus DNA and wild type P185BCR-ABL cDNAs cloned into the pAcC12 vector. Six days post-transfection,the cells were metabolically labeled with [³⁵S] methionine. The cellswere lysed and the lysates incubated with GST-GRB2 SH2 (lanes 1) or withanti-ABL pEX4 antibodies with protein A-Sepharose beads (lanes 2). Thebound proteins were analyzed by SDS 7% polyacrylamide gelelectrophoresis and fluorography.

FIG. 8B. Mutation of tyrosine 177 to phenylalanine in the BCR first exonabolishes binding of the GRB-2 SH2 domain to BCR-ABL. Sf9 insect cellswere co-transfected with wild type baculovirus DNA and P185 BCR-ABL(Y177F) cDNAs cloned into the pAcC12 vector. Six days post-transfection,the cells were metabolically labeled with [³⁵S] methionine. The cellswere lysed and the lysates incubated with GST-GRB2 SH2 (lanes 1) or withanti-ABL pEX4 antibodies with protein A-Sepharose beads (lanes 2). Thebound proteins were analyzed by SDS 7% polyacrylamide gelelectrophoresis and fluorography.

FIG. 9A. Formation of a BCR-ABL-GRB-2 complex in vivo requires thepresence of tyrosine 177 in the BCR first exon. COS cells weretransfected with P185 wild type (lanes 1-3) and P185 (Y177F) (lanes4-6), cDNAs cloned into the pSRα MSVtkneo vector. Three dayspost-transfection, the cells were lysed and the lysates incubated withnormal rabbit sera (NRS) (lanes 1 and 4) anti-ABL 2/3 rabbit antibodies(lanes 2 and 5) or anti-GRB-2 antibodies (lanes 3 and 6) for 2 hrs. at4° C. The immunoprecipitates were collected on protein A-Sepharose beadsand subjected to in vitro phosphorylation in the presence of [γ³²P]ATPand MnCl2. Proteins were analyzed by SDS 10% polyacrylamidegelelectrophoresis and autoradiography.

FIG. 9B. Formation of a BCR-ABL-GRB-2 complex in vivo requires thepresence of tyrosine 177 in the BCR first exon. Lysates of G418-selectedRat1 cells expressing P185 wild type (lanes 1-3) and P185 (Y177F) (lanes4-6) were incubated for 2 hrs. at 4° C. with anti-GRB-2 antibodies(lanes 1 and 4), anti-ABL 2/3 antibodies (lanes 2 and 5) or normalrabbit sera (NRS) (lanes 3 and 6). The immunoprecipitates were collectedon protein A-Sepharose beads. The bound proteins were analyzed by SDS10% polyacrylamide gel electrophoresis followed by immunoblotting withanti-ABL mouse monoclonal antibody. Immunoreactive bands were visualizedwith the ECL detection system (Amersham).

FIG. 10. Mutation of Tyrosine 177 in the BCR-ABL oncogene abolishes GRB2binding and decreases transforming capacity. Superscript legend:

a Names of the different P185 BCR-ABL forms. The mutated or deletedamino acids are indicated in parenthesis.

b Average frequency of colony formation in agar determined from twoplates per assay and four to five independent assays per construct.

c Cells were plated in agar 3 days after infection with helper-freeretroviral stocks.

d Cells were selected for 12 to 15 days with G418 (0.5 mg/ml) starting 3days after infection with helper-free retroviral stocks.

e Number of high density bone marrow cultures exhibiting transformedlymphoid outgrowth over the total of cultures plated. Data representthree separate experiments. In each experiment, cell cultures were setup after infection with the respective retroviral stock.

FIG. 11. Expression of wild type and mutant forms of BCR-ABL in Rat1cells. Rat1 cells were infected with retroviruses encoding for wild type(lane 1) and mutant (Y177F; lane 2) forms of P185 BCR-ABL or vectorcontrol (lane 3). Three days post-infection the cells were lysed andsubjected to Western blotting with anti-ABL mouse monoclonal antibody.Immunoreactive bands were visualized with the ECL detection system.

FIG. 12. Stimulation of transcriptional activation by BCR-ABL throughRas requires the presence of tyrosine 177 in the BCR first exon. NIH 3T3cells were transfected with 0.5 μg of the indicated BCR-ABL cDNA +/−5 μgH-Ras (17N) cDNA, along with the 1 μg pB4X-CAT reporter plasmid. At 48hr. post-transfection, cells were harvested and assayed for CAT activityas outlined in Section 9.1.1, below. Data were recorded in arbitraryunits; the basal signal due to activity of the reporter.

FIG. 13. Increase in Ras-GTP following infection of Rat1 cells withBCR-ABL retrovirus. Rat1 cells were infected with helper free retroviralstocks for P185 wild type (lane 1) or vector control (lane 2). Two dayspost infection, the cells were labeled with [³²P] orthophosphate (0.5mCi/ml) for 16 hours in phosphate-free medium. The cells were lysed andRas immunoprecipitated with anti-Ras monoclonal antibody. Guaninenucleotides bound to Ras were dissociated and subsequently separated bythin layer chromatography on PEI-cellulose plates in 0.75M KH2PO4, pH3.5. The position of GDP and GTP are indicated.

FIG. 14A. Rat1 cells expressing p210 BCR/ABL alone or also expressingwild type GRB2 or a truncated GRB2 were lysed and immunoprecipitatedwith anti-ABL antibody. Immunoprecipitates were subjected to an in vitroautokiase assay and the samples analyzed by SDS-PAGE (8% gel). No changein ABL kinase activity is seen in cells with elevated GRB2 expressioncompared to control.

FIG. 14B. Rat1 cells expressing p210 BCR/ABL alone or also expressingwild type GRB2 or a truncated GRB2 were lysed and proteins from thecleared cell lysate were electrophoretically separated using SDS-PAGE(15% gel). Separated proteins were electrophoretically transferred tonitrocellulose filters and then immunoblotted with anti-GRB2 Mab. AllGRB2 transformants express approximately the same levels of GRB2protein.

FIG. 15. Stimulation of transcriptional activation by BCR-ABL throughRas requires the presence of a GRB2 SH3 domain. Rat1 cells expressingp210 BCR/ABL cells were transfected with either of the GRB2 deletionmutants, along with the 1 μg pB4X-CAT reporter plasmid. At 48 hr.post-transfection, cells were harvested and assayed for CAT activity asoutlined in Section 9.1.1, below. Data were recorded in arbitrary units;the basal signal due to activity of the reporter.

FIG. 16. Lysates were prepared from different GRB2 expressing cell types(K562, Rat 1 cells transfected with v-raf, C-6 gliobastoma) and analyzedby immunoblotting with antibodies to GRB2. The multiple bands representGRB2 breakdown products.

5. DETAILED DESCRIPTION OF THE INVENTION

Described below are compositions and methods for the prevention andtreatment of cell proliferative disorders wherein a protein tyrosinekinase or a protein tyrosine phosphatase capable of complexing with anSH2- and/or SH3-containing member of the adaptor family of proteins isinvolved. Further, protein tyrosine kinase/adaptor protein complexes,protein tyrosine phosphatase/adaptor protein complexes, methods for theproduction of such complexes, and uses of these complexes are describedherein. Such uses may include, but are not limited to, theidentification of agents capable disrupting the interaction between thecomponents of such complexes.

This invention is based, in part, on the surprising discovery that theadaptor protein, GRB-2, binds the intracellular BCR-ABL product in vivo,is necessary for the activation of the oncogenic potential of theBCR-ABL product and that disruption of the GRB-2 signal transduction canreverse the transformed phenotype of cells and reduce tumor growth inanimals. The data representing this discovery is presented in theWorking Examples of Sections 6 through 12, below.

5.1. Protein Tyrosine Kinase/Adaptor Protein Complexes

The PTK/adaptor protein complexes of the invention comprise at least onemember of the PTK family of proteins and at least one member of theadaptor family of proteins, as described below. Under standardphysiological conditions, the components of such complexes are capableof forming stable, non-covalent attachments with one or more of theother PTK/adaptor protein complex components.

The PTK components of the PTK/adaptor protein complexes of the inventionare either intracellular, cytoplasmic non-receptor PTKs, intracellular,nuclear non-receptor PTKs, or transmembrane, receptor-type PTKs, each ofwhich comprises one or more characteristic peptide domains. Such domainsmay include one or more catalytic domains which may include, but are notlimited to, a tyrosine kinase domain. A tyrosine kinase catalytic domaingenerally ranges in length from about 250 to about 300 amino acids,corresponding to a molecular weight of approximately 30 kDa. Thelocation of the tyrosine kinase catalytic domain, while not fixed, isgenerally near the carboxyl terminus of the protein. Short, conserved,stretches of amino acid residues may be present within the tyrosinekinase domain, which alternate in sequence with variable-lengthstretches of amino acid residues which do not exhibit a high level ofconservation. The consensus sequences, corresponding to the most highlyconserved of the tyrosine kinase catalytic domain amino acid residueshave been compiled and are well known to those of ordinary skill in theart. See, for example, Hanks et al. (Hanks et al., 1991, Science241:42-52), and Wilks (Wilks, A. F., 1990, Prop. Growth Factor Res.2:97-111) which are incorporated herein, by reference, in theirentirety.; Among such consensus sequences are the PTK-specific sequencesD-L-R-A-A-N (SEQ ID NO:1) or D-L-A-A-R-N (SEQ ID NO:2), andP-I/V-K/R-W-T/M-A-P-E (SEQ ID NO:3). Moreover, see FIG. 1, for a diagramof additional examples of such sequence motifs.

The PTK component of the PTK/adaptor protein complexes of the inventionmay further include one or more non-catalytic domains, which mayinclude, but are not limited to, one or more SH2 and/or one or more SH3domains, one or more SH2-binding, and/or one or more SH3-binding peptidedomains, and/or (in the case of receptor PTKs) a hydrophobictransmembrane domain. An SH2 (i.e., src homology 2) non-catalytic domainis generally approximately 100 amino acid residues in length. Such SH2domains may contain a number of highly conserved or invariant amino acidresidues within several, preferably five, well-conserved amino acidsequence motifs, which are well known to those of ordinary skill in theart. See, for example Koch et al. (Koch et al., 1991, Science252:668-674), which is incorporated herein, by reference, in itsentirety. For example, the amino acid consensus sequences may include,but are not limited to, F-L-I-R-E-S (SEQ ID NO:4) and F-L-V-R-E-S (SEQID NO:5). The R residue of these consensus sequences is invariant amongSH2 domains. Such well-conserved amino acid sequences motifs areseparated by stretches of more variable amino acid sequence elements,which, while variable, generally contain one or more G or P residues.

An SH3 (i.e., src homology 3) non-catalytic domain is approximately 50amino acids residues in length. While the amino acid sequence within anSH3 domain may be variable, the 3-dimensional, tertiary, structure ofthe domain is well conserved. Such an SH3 tertiary structure is wellknown to those of ordinary skill in the art. See, for example, Koyama etal. (Koyama et al., 1993, Cell 72:945-952) which is incorporated herein,by reference, in its entirety.

SH2-binding peptide domains are well known in the art. See, for example,Songyang et al. (Songyang et al., 1993, Cell 72:767-778), Rotin et al.(Rotin et al., EMBO J. 11:559-567), and Skolnick et al. (Skolnick etal., 1993, EMBO J. 12:1929-1936), which are incorporated herein, byreference, in its entirety. SH2 domains may exhibit a specificity forcertain SH2-binding domains. For example, SH2-binding peptide domainsmay include, but are not limited to aphosphotyr-hydrophilic-hydrophilic-Ile/Pro amino acid sequence motif(generally,, such a sequence motif is preferred for SH2 domains of thetype found in, for example, the src, fyn, lck, fgr, abl, crk, and nckproteins), and phosphoTyr-hydrophobic-X-hydrophobic, and/orphosphotyr-Met-X-Met (generally, such sequence motifs are preferred forSH2 domains of the type found in, for example, p85, phospholipase C-γ,and SHPTP2 proteins). Further, a consensus sequence developed from theanalysis of the domains of several proteins that bind the SH2 domains ofthe GRB-2 protein has been determined to be X-P-X-Y-V/I-N-V/I (SEQ IDNO:6). In addition, SH2-binding peptide domains may comprise regionsrich in Ser and Thr residues some or all of which are phosphorylated(Pendergast et al., 1991, Cell 66:161-171).

SH3-binding peptide domains are also well known to those of ordinaryskill in the art. See, for example, Ren et al. (Ren et al., 1993,Science 259:1157-1161) and Cicchetti et al. (Cicchetti et al., 1992,Science 257:803-806), which are incorporated herein, by reference, intheir entirety. Such SH3-binding peptide domains are generally rich inPro amino acid residues, although amino acid residues in addition tosolely Pro are also critical for SH3 binding. One possible consensussequence for a SH3-binding domain is: X-P-X-X-P-P-P-hydrophobicresidue-X-P (SEQ ID NO:7). Further, the SH3 domains of GRB-2 have beendetermined to be P-P-P-V-P-P-R-R (SEQ ID NO:8), an amino acid sequencemotif found in the SOS protein (Li et al., 1993, Nature 363:8588;Schlessinger, 1993, TIBS, 18:273-275.

Intracellular, cytoplasmic PTK components of the PTK/adaptor proteincomplex of the invention may include, for example, members of the Srcfamily, such molecules as src, yes, fgr, fyn, lyn, hck, lck, and blk;members of the Fes family, such as fes and fer; members of the Ablfamily, such as abl and arg; and members of the Jak family, such as jak1and jak2. In a preferred embodiment of the invention, the PTK componentof the PTK/adaptor protein complex is the. intracellular PTK product ofthe BCR-ABL gene. Transmembrane, receptor PTK components of thePTK/adaptor protein complex of the invention may include, for example,such molecules as members of the FGF receptor, Sevenles/ROS, Insulinreceptor, PDGF receptor, EGF receptor family of growth factor receptorsor any other molecule that associates with he adaptor protein (see forexample Lowenstein et al., 1992, Cell 70:431-442).

The adaptor protein components of the PTK/adaptor protein complexes ofthe invention comprise one or more SH2 and/or one or more SH3non-catalytic domains. The SH2 and SH3 domains which may be a part ofthe adaptor proteins are as described, above, for the PTK components.Adaptor proteins which may be components of the PTK/adaptor proteincomplexes of the invention, may include, for example, p85, c-Crk, SHC,Nck, ISGF3α, guanine triphosphatase activator protein (GAP), and membersof the GRB subfamily of proteins, such as GRB1, GRB-2, GRB-3, GRB-4,GRB-7, and GRB-10. In a preferred embodiment, the PTK/adaptor proteincomplex of the invention comprises the PTK product of the BCR-ABL geneand GRB-2 protein.

The complexes of the invention, and/or the individual components of thecomplexes of the invention may be substantially purified utilizingmethods which are described below, in Section 5.3.1. Further, the PTKand/or adaptor components of the complexes of the invention may beproduced by utilizing a variety of methods which include, but are notlimited to, chemical synthesis or recombinant DNA techniques, asdescribed in Section 5.3.2, below.

5.2. Protein Tyrosine Phosphatase/Adaptor Protein Complexes

The PTP/adaptor protein complexes of the invention comprise at least onemember of the PTP family of proteins and at least one member of theadaptor family of proteins. Under standard physiological conditions, thecomponents of such complexes are capable of forming stable, non-covalentattachments with one or more of the other PTP/adaptor protein complexcomponents.

The PTP components of the PTP/adaptor protein complexes of the inventionare either cytoplasmic, intracellular, non-receptor PTPs ortransmembrane, receptor-type PTPs, each of which comprises one or morecharacteristic peptide domains. Such domains may include one or morecatalytic domains which may include, but are not limited to, a tyrosinephosphatase domain. Generally, a tyrosine phosphatase catalytic domainis approximately about 230 amino acids in length. Approximately 40 ofthe amino acid residues of the catalytic phosphatase domain are highlyconserved, and of these, a very highly conserved segment of 11 aminoacid residues with the consensus sequence I/V-H-C-X-A-G-X-X-R-S/T-G (SEQID NO:9) is generally present. Non-receptor PTPs generally contain asingle such catalytic domain, while the transmembrane receptor PTPsgenerally contain two such catalytic domains separated by a peptidesegment approximately 58 amino acid residues in length.

The PTP component of the PTP/adaptor protein complexes of the inventionmay further include one or more non-catalytic domains, which mayinclude, but are not limited to one or more SH2 domains, one or more SH3domains, one or more SH2-binding domains, and/or one or more SH3-bindingdomains. Each of these non-catalytic domains may be as described, above,in Section 5.1.

Transmembrane, receptor PTP components of the PTP/adaptor proteincomplexes of the invention may include, for example, CD45, LAR, RPTPα,RPTPβ, RPTPχ, and RPTPκ. Intracellular, cytoplasmic PTP components ofthe PTP/adaptor protein complexes of the invention may include, forexample, PTP1C, PTP1D, and corkscrew.

5.3. Purification and Production of PTK/Adaptor and PTP/AdaptorComplexes

5.3.1. Purification Methods

The PTK/adaptor and PTP/adaptor complexes of the invention may besubstantially purified, i.e., may be purified away from at least 90% (ona weight basis), and from at least 99%, if desired, of other proteins,glycoproteins, and other macromolecules with which it is associated.Such purification can be achieved by utilizing a variety of procedureswell known to those of skill in the art, such as subjecting cells,tissue or fluid containing the PTK/adaptor or PTP/adaptor complex to acombination of standard methods, for example, ammonium sulfateprecipitation, molecular sieve chromatography, and/or ion exchangechromatography. Alternatively, or additionally, a PTK/adaptor orPTP/adaptor complex may be purified by immunoaffinity chromatographyusing an immunoadsorbent column to which an antibody is immobilizedwhich is capable of binding to one or more components of the PTK/adaptoror PTP/adaptor complex. Such an antibody may be of monoclonal orpolyclonal in origin. Other useful types of affinity purification for aPTK/adaptor or PTP/adaptor complex may utilize, for example, asolid-phase substrate which binds the catalytic domain (i.e., kinasedomain of PTK or phosphatase domain of PTP), or an immobilized bindingsite for noncatalytic domains of the PTK, PTP, and/or adaptor componentsof the complex, which bind in such a manner as to not disrupt thecomplex.

The PTK/adaptor or PTP/adaptor complexes of the present invention may bebiochemically purified from a variety of cell or tissue sources. Forpurification of a naturally occurring PTK/adaptor complex, cellularsources may include, for example, baculovirus-infected SF9 cells, A-431,CHO, and/or 3T3 cells. In a preferred embodiment of the presentinvention, the PTK/adaptor complex comprises a BCR-ABL PTK and a GRB2protein. Sources for the purification of such a PTK/GRB-2 complex mayinclude, but are not limited to K562, NMG-01, and ALL-1 cell lines.

5.3.2. Synthesis Methods

Methods for the synthesis of polypeptides or fragments thereof, whichare capable of acting as components of the PTK/adaptor or PTP/adaptorcomplexes of the present invention, are well-known to those of ordinaryskill in the art. See, for example, Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman and Co., N.Y., whichis incorporated herein, by reference, in its entirety. Components of aPTK/adaptor or PTP/adaptor complexes which have been separatelysynthesized or recombinantly produced, may be reconstituted to form acomplex by standard biochemical techniques well known to those skilledin the art. For example, samples containing the components of thePTK/adaptor complex may be combined in a solution buffered with greaterthan about 150 mM NaCl, at a physiological pH in the range of 7, at roomtemperature. For example, a buffer comprising 20 mM Tris-HCl, pH 7.4,137 mMNaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholateand 2 mM EDTA could be used. Such a procedure may also be utilized forthe reconstitution of a PTP/adaptor complex.

Methods for preparing the components of PTK/adaptor complexes of theinvention by expressing nucleic acid encoding PTK, PTP, and/or adaptorproteins are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining PTK, PTP, and/or adaptor protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. DNA and RNAsynthesis may, additionally, be performed using an automatedsynthesizers. See, for example, the techniques described in Maniatis etal., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. and Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,N.Y.

A variety of host-expression vector systems may be utilized to expressthe coding sequences of the components of the PTK/adaptor PTP/adaptorcomplexes of the invention. These include but are not limited to,microorganisms such as bacteria (e.g. E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing PTK, PTP, or adaptor protein coding sequences; yeast(e.g. Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the PTK, PTP, and/or adaptor proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g. baculovirus) containing the PTK, PTP, and/oradaptor protein coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g. cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g. Ti plasmid) containing the PTK, PTP, and/oradaptor protein coding sequences coding sequence; or mammalian cellsystems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g. metallothionein promoter) or from mammalian viruses (e.g.the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thePTK/adaptor or PTP/adaptor complex being expressed. For example, whenlarge quantities of PTK/adaptor or PTP/adaptor complex proteins are tobe produced for the generation of antibodies or to screen peptidelibraries, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the PTK, PTP,and/or adaptor protein coding sequence may be ligated individually intothe vector in frame with the lac Z coding region so that a fusionprotein is produced; pIN vectors (Inouye et al., 1985, Nucleic AcidsRes. 13:3101-3109; Van-Heeke et al., 1989, J. Biol. Chem.264:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption to glutathione-agarose beadsfollowed by elution in the presence of free glutathione. The PGEXvectors are designed to include thrombin or factor Xa protease cleavagesites so that the cloned PTK and/or adaptor protein can be released fromthe GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The PTK/adaptor or PTP/adaptor complexcomponent coding sequences may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of the coding sequences will result in inactivationof the polyhedrin gene and production of non-occluded recombinant virus(i.e., virus lacking the proteinaceous coat coded for by the polyhedringene). These recombinant viruses are then used to infect Spodopterafrugiperda cells in which the inserted gene is expressed. (e.g. seeSmith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the PTK/adaptor or PTP/adaptor complex component codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g. the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g. region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing PTK, PTP, and/or adaptorproteins in infected hosts. (e.g. see Logan et al., 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Specific initiation signals may also berequired for efficient translation of inserted PTK, PTP, and/or adaptorcoding sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire PTK, PTP, or adaptorprotein gene, including its own initiation codon and adjacent sequences,is inserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the PTK, PTP, or adaptor coding sequence is inserted,exogenous translational control signals, including the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.glycosylation) and processing (e.g. cleavage) of protein products may beimportant for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. Forlong-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably coexpressboth the PTK and adaptor protein or PTP and adaptor protein may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with the PTK andadaptor protein DNA or PTP and adaptor protein DNA independently orcoordinately controlled by appropriate expression control elements (e.g.promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker.: Following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines which coexpress both the PTK and adaptorprotein or PTP and adaptor protein. Such engineered cell lines areparticularly useful in screening and evaluation of compounds that affectsignals mediated by the complexes.

A number of selection systems may be used, including but not limited tothe herpes simplex virus. thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska etal., 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147) genes.

New members of the PTK, PTP, and/or adaptor protein families capable offorming the complexes of the invention may be identified and isolated bymolecular biological techniques well known in the art. For example, apreviously unknown PTK, PTP, or adaptor protein gene may be isolated byperforming a polymerase chain reaction (PCR) using two degenerateoligonucleotide primer pools designed on the basis of highly conservedsequences within domains common to members of the PTK, PTP, or adaptorprotein family. The template for the reaction may be cDNA obtained byreverse transcription of mRNA prepared from cell lines or tissue knownto express PTK/adaptor and/or PTP/adaptor complexes. The PCR product maybe subcloned and sequenced to insure that the amplified sequencesrepresent the sequences of a member of the PTK, PTP, or adaptorsubfamily. The PCR fragment may then be used to isolate a full lengthPTK, PTP, or adaptor protein CDNA clone by radioactively labeling theamplified fragment and screening a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary. For a review of cloning strategies which may be used, see e.g.Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold SpringsHarbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols inMolecular Biology, (Green Publishing Associates and Wiley Interscience,N.Y.).

A general method for cloning previously unknown adaptor proteins hasbeen described by Skolnick (Skolnick, E. Y., 1991, Cell 65:75) andSkolnick et al., (U.S. patent application Ser. No. 07/643,237) which areincorporated herein, by reference, in their entirety. Briefly, newmembers of the adaptor family of proteins may be identified by theirability to specifically bind to at least a portion of atyrosine-phosphorylated peptide comprising an adaptor-protein-bindingregion. Such a region may include, but is not limited to an SH2-bindingdomain.

5.4. Derivatives of PTK/adaptor and PTP/adaptor Complexes

Also provided herein are functional derivatives of a PTK/adaptor andPTP/adaptor complexes. By “functional derivative” is meant a “chemicalderivative,” “fragment,” “variant,” “chimera,” or “hybrid” of thePTK/adaptor and PTP/adaptor complex, which terms are defined below. Afunctional derivative retains at least a portion of the function of thePTK, PTP, or adaptor protein, for example reactivity with an antibodyspecific for the PTK/adaptor or PTP/adaptor complex, PTK or PTPenzymatic activity or binding activity mediated through noncatalyticdomains, which permits its utility in accordance with the presentinvention.

A “chemical derivative” of the PTK/adaptor or PTP/adaptor complexcontains additional chemical moieties not normally a part of theprotein. Covalent modifications of the protein complex or peptides areincluded within the scope of this invention. Such modifications may beintroduced into the molecule by reacting targeted amino acid residues ofthe peptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues, as describedbelow.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted. with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect or reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(α)of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineε-amino group.

Tyrosyl residues are well-known targets of modification for introductionof spectral labels by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction carbodiimide (R′-N-C-N-R′) such as1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residue are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful, for example, forcross-linking the component peptides of the PTK/adaptor or PTP/adaptorcomplexes to each other or the PTK/adaptor receptor complex to awater-insoluble support matrix or to other macromolecular carriers.Commonly used cross-linking agents include, for example,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (Creighton, T. E., Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility,absorption, biological half life, and the like. The moieties mayalternatively eliminate or attenuate any undesirable side effect of theprotein complex and the like. Moieties capable of mediating such effectsare disclosed, for example, in Remington's Pharmaceutical Sciences,1990, 18th ed., Mack Publishing Co., Easton, Pa.

The term “fragment” is used to indicate a polypeptide derived from theamino acid sequence of the PTK, PTP, or adaptor proteins, of thePTK/adaptor or PTP/adaptor complexes having a length less than thefull-length polypeptide from which it has been derived. Such a fragmentmay, for example, be produced by proteolytic cleavage of the full-lengthprotein. Preferably, the fragment is obtained recombinantly byappropriately modifying the DNA sequence encoding the PTK, PTP, oradaptor proteins to delete one or more amino acids at one or more sitesof the C-terminus, N-terminus, and/or within the native sequence.Fragments of a PTK, PTP, or adaptor protein, when present in a complexresembling the naturally occurring PTK/adaptor or PTP/adaptor complex,are useful for screening for compounds that act to modulate signaltransduction, as described below. It is understood that such fragments,when present in a complex may retain one or more characterizing portionsof the native PTK/adaptor or PTP/adaptor complex. Examples of suchretained characteristics include: catalytic activity; substratespecificity; interaction with other molecules in the intact cell;regulatory functions; or binding with an antibody specific for thenative PTK/adaptor or PTP/adaptor complex, or an epitope thereof.

Another functional derivative intended to be within the scope of thepresent invention is a PTK/adaptor or PTP/adaptor complex comprising atleast one “variant” polypeptide (e.g. PTK, PTP, or adaptor) which eitherlack one or more amino acids or contain additional or substituted aminoacids relative to the native polypeptide. The variant may be derivedfrom a naturally occurring PTK/adaptor or PTP/adaptor complex componentby appropriately modifying the PTK, PTP, and/or adaptor protein DNAcoding sequence to add, remove, and/or to modify codons for one or moreamino acids at one or more sites of the C-terminus, N-terminus, and/orwithin the native sequence. It is understood that such variants havingadded, substituted and/or additional amino acids retain one or morecharacterizing portions of the native PTK/adaptor or PTP/adaptorcomplex, as described above.

A functional derivative of PTK/adaptor or PTP/adaptor complexescomprising PTK, PTP, and/or adaptor proteins with deleted, insertedand/or substituted amino acid residues may be prepared using standardtechniques well-known to those of ordinary skill in the art. Forexample, the modified components of the functional derivatives may beproduced using site-directed mutagenesis techniques (as exemplified byAdelman et al., 1983, DNA 2:183) wherein nucleotides in the DNA codingthe sequence are modified such that a modified coding sequence ismodified, and thereafter expressing this recombinant DNA in aprokaryotic or eukaryotic host cell, using techniques such as thosedescribed above. Alternatively, components of functional derivatives ofPTK/adaptor or PTP/adaptor complexes with amino acid deletions,insertions and/or substitutions may be conveniently prepared by directchemical synthesis, using methods well-known in the art. The functionalderivatives of the PTK/adaptor or PTP/adaptor complexes typicallyexhibit the same qualitative biological activity as the nativecomplexes.

5.5. Antibodies to PTK/Adaptor or PTP/Adaptor Complexes

The present invention further relates to antibodies which are capable ofspecifically recognizing a PTK/adaptor complex or PTP/adaptor complex oran epitope thereof, or of specifically recognizing an epitope on eitherthe PTK, PTP, or adaptor components of the complex which would not berecognized by the antibody when the PTK, PTP, and/or adaptor componentis present separate and apart from the PTK/adaptor or PTP/adaptorcomplex. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a FAb expression library, anti-idotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a PTK/adaptorcomplex in a biological sample, or, alternatively, as a method for theinhibition of PTK/adaptor complex formation, thus, inhibiting thedevelopment of a cell proliferative disorder.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as PTK/adaptor complex, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, various hostanimals may be immunized by injection with the PTK/adaptor orPTP/adaptor complex including but not limited to rabbits, mice, rats,etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

A monoclonal antibody, which is a substantially homogeneous populationof antibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to thehybridoma technique of Kohler et al. (Kohler et al., Nature 256:495-497(1975) and U.S. Pat. No. 4,376,110), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. Production of high titers of mAbs invivo production makes this the presently preferred method. ofproduction.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively,. techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce PTK/adaptorcomplex-specific or PTP/adaptor complex-specific single-chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragment of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

Antibody fragments which contain specific binding sites of a PTK/adaptoror PTP/adaptor complex may be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity to the PTK/adaptor or PTP/Adaptorcomplex.

5.6. Treatment of PTK/Adaptor Protein- and PTP/Adaptor Protein-RelatedCell Proliferative Disorders

The present invention demonstrates, for the first time, that the bindingof a member of the SH2- and/or SH3-containing family of adaptor proteinscan represent an essential step in oncogenesis and the transformationprocess. More specifically, the data presented in the Working Examplesin Section 6 through 12, below, detail the binding of the GRB2 member ofthe GRB subfamily of adaptor proteins to the intracellular PTK productof the human BCR-ABL gene.

Described in this Section are some of the variety of uses to which thebinding of such PTKs and adaptor proteins and/or PTPs and adaptorproteins can be put for the treatment of cell proliferative disordersinvolving such complexes. The uses described herein focus on, but arenot limited to, the identification of agents capable of disrupting suchcomplexes (i.e., decreasing or inhibiting the interaction between thecomponent PTK or PTP, and adaptor members of the complexes), and theutilization of such compounds for the treatment of cell proliferativedisorders involving a PTK or a PTP capable of complexing with a memberof the SH2- and/or SH3-containing family of adaptor proteins.“Disrupting”, s used herein, is meant to refer not only to a physicalseparation of protein complex components, but also refers to aperturbation of the activity of the complexes, regardless of whether ornot such complexes remain able, physically, to form. “Activity”, as usedhere, refers to the function of the protein complex in the signaltransduction cascade of the cell in which such a complex is formed,i.e., refers to the function of the complex in effecting or inhibitingtransduction of an extracellular signal into a cell. Examples of suchcell proliferative disorders, include, but are not limited to, oncogenicdisorders such as, for example. chronic myelogenous and acutelymphocytic leukemias as well as psoriasis and atherosclerosis. Thecomplexes of the present invention may also be involved in such cellularprocesses such as activation, differentiation, and survival.

Depending on the individual PTK/adaptor protein complex or PTP/adaptorprotein complex, disrupting the interaction between component members ofsuch complexes may have differing modulatory effects on the signaltransduction event involved, i.e., the effect of the complex disruptionmay activate, reduce, or block the signal normally transduced into thecell. Likewise, depending on the cell proliferative disorder involved,either activation, reduction, or blockage of the signal normallytransduced into the cell will be desirable for the treatment of thedisorder. For example, one effect of the complexing of the BCR-ABL PTKwith the GRB-2 adaptor protein causes the activation of the Rassignalling pathway (see the Working Example presented, below, in Section8). Thus, the disruption of such a BCR-ABL PTK/GRB-2 adaptor proteincomplex would inhibit the transduction of the abnormal signal andprevent activation of the Ras pathway. Alternatively, a cellproliferative disorder involving a PTK/adaptor or PTP/adaptor complexmay, for example, develop because the presence of such complexes bringsabout the aberrant inhibition of a normal signal transduction event. Insuch a case, the disruption of the complex would allow the restorationof the usual signal transduction event. Further, an aberrant complex maybring about an altered subcellular adaptor protein localization, whichmay result in, for example, such dysfunctional cellular events as acytoskeletal reorganization, as can be the case for the GRB-2 member ofthe GRB subfamily of adaptor proteins. An inhibition of the PTK/adaptoror PTPladaptor complex in this case would allow for restoration ormaintenance of a normal cellular architecture. Still further, an agentor agents that cause(s) disruption of the PTK/adaptor or PTP/adaptorcomplex may bring about the disruption of the interactions among otherpotential components of such a complex, which may include, but are notlimited to an SOS protein.

When considering PTK/adaptor and PTP/adaptor protein complexes whereinthe PTK or the PTP component of the complex is a transmembrane,receptor-type PTK or PTP molecule, the receptors or their ligands may beused directly to modulate signal transduction events which may lead tothe development of cell proliferative disorders. For example, taking thecase of PTKs, soluble PTKs, peptides representing extracellular PTKdomains, or peptides representing those portions of extracellular PTKdomains which are known to bind ligands may be administered, usingtechniques well known to those skilled in the art, that, when exposed tothe PTK-expressing cells of interest could act to compete withendogenous transmembrane PTK receptor molecules for available ligands,thus reducing or inhibiting ligand binding to endogenous PTKs. Theeffect of such a procedure could bring about a reduction or inhibitionof the interaction between the PTK and the adaptor protein, possibly byblocking the autophosphorylation of the PTK which could, in turn, reducethe affinity of the adaptor protein for the PTK molecule. An analogoussituation would hold in the case of PTPs.

In addition, again when considering receptor-type PTKs, extracellularmolecules which bind to such PTKs may be administered, using techniqueswell known to those in the art, which, while binding the PTK do notactivate the molecule. Extracellular molecules of this type may becomposed, for example, of modified forms of a native ligand for the PTKof interest, such that receptor binding may still occur, but activationof the kinase does not. A molecule with such a design could act in muchthe same way that administration of soluble PTK would, in that bothprocedures could have the final effect of reducing or inhibiting theformation of the PTK/adaptor protein complexes. Once again, an analogoussituation would occur in the case of the PTPs.

Still further, molecules which are capable of binding native ligands ofthe receptor PTKs of the PTK/adaptor complexes or the receptor PTPs ofthe PTP/adaptor complexes of the invention may be administered, usingtechniques well known to those of skill in the art. Molecules in thisclass would act to inhibit the ligands' ability to bind it's respectivereceptor, and thus would have the final effect of reducing or inhibitingthe formation of PTK/adaptor or PTP/adaptor protein complexes.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transmucosal, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, just to name a few. For injection, the agents ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For such transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Agents which act intracellularly to directly interfere with theformation of the PTK/adaptor and/or PTP/adaptor complexes of theinvention may be administered for the treatment of cell proliferativedisorders. Such agents may include, but are not limited to, peptidesand/or phosphopeptides comprising SH2 and or SH3 domains, or SH2 and/orSH3-binding domains, small organic molecules or extracts of naturalproducts which would act to compete with the components of the complexesfor binding, thus reducing or inhibiting the formation of complexes,which would, in turn, reduce or inhibit the development of the cellproliferative disorder of interest. SH2 and SH3 peptide domains, andSH2-binding and SH3-binding peptide domains are as described, above, inSection 5.1. Such agents may also disrupt the complex by interferingwith down stream signaling capability instead of or in addition tocomplex formation.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. Liposomes are spherical lipid bilayerswith aqueous interiors. All molecules present in an aqueous solution atthe time of liposome formation are incorporated into the aqueousinterior. The liposomal contents are both protected from the externalmicroenvironment and, because liposomes fuse with cell membranes, areefficiently delivered into the cell cytoplasm. Additionally, due totheir hydrophobicity, small organic molecules may be directlyadministered intracellularly.

Nucleotide sequences encoding the peptide agents which are to beutilized intracellularly may be expressed in the cells of interest,using techniques which are well known to those of ordinary skill in theart. For example, expression vectors derived from viruses such asretroviruses, vaccinia virus, adeno-associated virus, herpes viruses, orbovine papilloma virus, may be used for delivery and expression of suchnucleotide sequences into the targeted cell population. Methods for theconstruction of such vectors are well known. See, for example, thetechniques described in Maniatis et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y.

Alternatively, antibodies capable of interfering with PTK/adaptor and/orPTP/adaptor complex formation may be administered for the treatment ofcell proliferative disorders involving a PTK or PTP capable of forming acomplex with an adaptor protein. For example, neutralizing antibodieswhich are capable of interfering with ligand binding to receptor typePTKs or PTPs may be administered using techniques such as thosedescribed above. The effect of such an administration would be similarto that described, above, for the administration of soluble PTKs orPTPS. Additionally, neutralizing antibodies which bind to intracellularepitopes to effect a disruption of PTK/adaptor or PTP/adaptor complexformation may also be administered. Such antibodies may be administered,for example, by utilizing the techniques described above for theadministration of agents intended to act intracellularly. Alternatively,nucleotide sequences encoding single-chain antibodies may be expressedwithin the target cell population by utilizing, for example, techniquessuch as those described in Marasco et al. (Marasco et al., 1993, Proc.Natl. Acad. Sci. USA 90:7889-7893).

The PTK/adaptor complexes and/or PTP/adaptor complexes of the inventionmay be used to screen for additional molecules that can act to disruptthe activity of the component members of such complexes, and thus may becapable of modulating the signal transduction event such complexeseffect. Such compounds may include, but are not limited to, peptidesmade of D- and/or L-configuration amino acids (in, for example, the formof random peptide libraries; see Lam et al., 1991, Nature 354:82-84),phosphopeptides (in, for example, the form of random or partiallydegenerate, directed phosphopeptide libraries, see Songyang et al.,1993, Cell 767-778), antibodies, and small organic molecules.

For example, compounds that bind to individual components, or functionalportions of the individual components of the PTK/adaptor or PTP/adaptorcomplexes (and may additionally be capable of disrupting complexformation) may be identified. A functional portion of an individualcomponent of the complexes may be defined here as a peptide portion ofan individual component of a complex still capable of forming a stablecomplex with another member of the complex. For example, a peptideportion of the SH2-binding domain of a PTK which is still capable ofstably binding an SH2 domain of an adaptor protein, and thus is stillcapable of forming a complex with that adaptor protein. Further, in thecase of the catalytic domains of the individual PTK or PTP components ofthe invention, a functional portion of a catalytic domain may refer to apeptide still capable of stably binding a substrate molecule.

One method utilizing this approach that may be pursued in the isolationof such PTK/adaptor or PTP/adaptor complex component-binding moleculeswould include the attachment of a component molecule, or a functionalportion thereof, to a solid matrix, such as agarose or plastic beads,microtiter wells, petri dishes, or membranes composed of, for example,nylon or nitrocellulose, and the subsequent incubation of the attachedcomponent molecule in the presence of a potential component-bindingcompound or compounds. After incubation, unbound compounds are washedaway, component-bound compounds are recovered. By utilizing thisprocedure, large numbers of types of molecules may be simultaneouslyscreened for PTK/adaptor or PTP/adaptor complex component-bindingactivity.

The PTK/adaptor complex components which may be utilized in the abovescreening method may include, but are not limited to, PTK molecules orfunctional portions thereof, such as PTK catalytic domains,phosphorylation domains, SH2 domains, SH3 domains, SH2-binding domains,or SH3-binding domains, and adaptor proteins, or functional portionsthereof, such as SH2 domains and SH3 domains. The peptides used may bephosphorylated, e.g. may contain at least one phosphorylated amino acidresidue, preferably a phosphorylated Tyr amino acid residue, or may beunphosphorylated. A phosphorylation domain may be defined as a peptideregion that is specifically phosphorylated at certain amino acidresidues. A functional portion of such a phosphorylation domain may bedefined as a peptide capable of being specifically phosphorylated atcertain amino acids by a specific PTK. A functional portion of an SH2domain may be defined as a peptide comprising at least a portion of anSH2 domain which is capable of specifically binding an SH2-bindingdomain. Likewise, a functional portion of an SH3 domain may be definedas a peptide comprising at least a portion of an SH3 domain which iscapable of specifically binding an SH3-binding domain. A functionalportion of an SH2-binding domain may be defined as a peptide capable ofbinding an SH2 domain, and may be at least about 4 amino acid residuesin length. A functional portion of an SH3-binding domain may be definedas a peptide capable of binding an SH3 domain, and may be at least about4 amino acids in length, with a length of about 1 amino acid residuesbeing preferred.

The PTP/adaptor complex components which may be utilized in the abovescreening method may include, but are not limited to, PTP molecules orfunctional portions thereof, such as PTP catalytic domains,phosphorylation domains, SH2 domains, SH3 domains, SH2-binding domains,or SH3-binding domains, and adaptor proteins, or functional portionsthereof, such as SH2 domains and SH3 domains. The peptides used may bephosphorylated, e.g. may contain at least one phosphorylated amino acidresidue, preferably a phosphorylated Tyr amino acid residue, or may beunphosphorylated. A phosphorylation domain may be defined as a peptideregion that is specifically phosphorylated at certain amino acidresidues. A functional portion of such a phosphorylation domain may bedefined asia peptide capable of being specifically phosphorylated atcertain amino acids by a specific PTK.Functional portions of SH2, SH3,SH2-binding, and SH3-binding domains may be as described above.

Molecules exhibiting binding activity may be further screened for anability to disrupt PTK/adaptor or PTP/adaptor complexes. Alternatively,molecules may be directly screened for an ability to disrupt PTK/adaptoror PTP/adaptor complexes. For example, in vitro complex formation may beassayed by, first, immobilizing one component, or a functional portionthereof, of the complex of interest to a solid support. Second, theimmobilized complex component may be exposed to a compound such as oneidentified as above, and to the second component, or a functionalportion thereof, of the complex of interest. Third, it may be determinedwhether or not the second component is still capable of forming acomplex with the immobilized component in the presence of the compound.

Additionally, in vivo complex formation may be assayed by utilizingco-immunoprecipitation techniques well known to those of skill in theart. Briefly, a cell line capable of forming a PTK/adaptor orPTP/adaptor complex of interest may be exposed to a compound such as oneidentified as above, and a cell lysate may be prepared from this exposedcell line. An antibody raised against one of the components of thecomplex of interest may be added to the cell lysate, and subjected tostandard immunoprecipitation techniques. In cases where a complex isstill formed, the immunoprecipitation will precipitate the complex,whereas in cases where the complex has been disrupted, only the complexcomponent to which the antibody is raised will be precipitated.

The effect of an agent on the transformation capability of thePTK/adaptor or PTP/adaptor complex of interest may be directly assayed.Such agents may but are not required to, include those agents identifiedby utilizing the above screening technique. For example, an agent oragents may be administered of a cell such as a fibroblast orhematopoietic cell capable of forming a PTK/adaptor complex which, inthe absence of any inhibitory agent, would lead to the cell'stransformation (Muller et al., 1991, Mol. Cell. Biol. 11:1785-1792;McLaughlin et al.,, 1987, Proc. Natl. Acad. Sci. USA 84:6558-6562). Thetransformation state of the cell may then be measured in vitro, bymonitoring, for example, its ability to form colonies in soft agar (Lugoet al., 1989, Mol. Cell. Biol. 9:1263-1270; Gishizky et al., 1992,Science 256:836-839). Alternatively, a cell's transformation state maybe monitored in vivo by determining its ability to form tumors inimmunodeficient nude on severe combined immunodeficiency (SCID) mice(Sawyers et al., 1992, Blood 79:2089-2098). Further, in the case ofBCR-ABL, an agent or agents may be administered to animal models of ALLand/or CML which are well known to those of ordinary skill in the art(Gishizky et al., 1993, Proc. Natl. Acad. Sci. USA 90:3755-3759) and/orreverse the progress of this oncogenic disorder.

Agents capable of disrupting PTK/adaptor and/or PTP/adaptor complexformation and capable of reducing or inhibiting cell; proliferativedisorders which arise from the formation of such complexes may be usedin the treatment of patients exhibiting or at risk for such disorders. Asufficient concentration of an agent or agents such as those describedabove may be administered to a patient so that the cell proliferativecapability of cells which, in the absence of such agents, would containPTK/adaptor and/or PTP/adaptor protein complexes, is reduced oreliminated.

Alternatively, in the case of hematopoietic cell proliferativedisorders, such as leukemias, rather than direct administration to thepatient, the agent or agents may be used in conjunction with autologousbone marrow transplantation and chemoradiotherapy techniques, which arewell known to those of skill in the art. Briefly, an aliquot of bonemarrow cells, generally taken from the pelvis, are removed from thepatient. The cells are then cultured in the presence of a concentrationof agent or agents which is capable of effectively disruptingPTK/adaptor or PTP/adaptor complex. By blocking the signal transductionpathway of those bone marrow cells capable of forming such complexes,one selects against the presence of clonal descendants of these cells,thus effectively purging the cultures of those cells responsible for thehematopoietic cell proliferative disorder being treated. While the bonemarrow cells are being cultured and purged of cells with a highoncogenic capacity, the patient is treated with chemoradiotherapyappropriate for the disease involved, using techniques and doses wellknown to those of skill in the art. Upon completion of suchchemoradiotherapy treatment, the patient receives an autologous infusionof the cultured bone marrow cells, which have been purged of oncogeniccells.

In a preferred embodiment of the invention, a PTK/adaptor complex isdisrupted or prevented and is one in which the PTK component is anintracellular PTK product of the human BCR-ABL gene, and the adaptorprotein component is a GRB-2 member of the GRB subfamily of adaptorproteins. Further, the cell proliferative disorders which administrationof such agents treats, in this preferred embodiment include, but are notlimited to, chronic myelogenous leukemia and acute lymphocytic leukemia.

6. EXAMPLE

BCR-ABL Associates with GRB-2 Both in vitro and in vivo

In the Working Example presented in this section, it is demonstratedthat the GRB-2 member of the GRB subfamily of adaptor proteins binds tothe BCR-ABL intracellular PTK both in vitro and in vivo.

6.1. Materials and Methods

6.1.1 Cells and Viruses

Spodoptera frugiperda (Sf9) insect cells were propagated in completeGrace's media (Smith, G. E., 1983, Mol. Cell. Biol. 3:2156-2165). ThePh¹-positive leukemic cell lines K562 and NMG-01 were derived frompatients with chronic myelogenous leukemia and express P210 BCR-ABL. ThePH¹-positive cell line, ALL-1, was derived from a patient with acutelymphocytic leukemia and expresses P185 BCR-ABL. The PH¹-positive celllines were cultured in RPMI 1640 medium with 10% fetal calf serum. COSAfrican green monkey cells and Rat1 fibroblasts were grown in DMEMmedium with 5% fetal calf serum.

Recombinant baculoviruses for expression of cBCR, cABL and BCR-ABL wereprepared by cotransfecting the corresponding cDNAs cloned into thepAcCI2 baculovirus vector in the presence of wild type baculovirus DNAas described (Pendergast et al., 1991, Cell 66:161-171; Pendergast etal., 1991, Proc. Natl. Acad. Sci. USA 88:5927-5931).

Helper-free retroviral stocks were prepared by transient hyperexpressionin COS cells according to methods previously described (Muller et al.,1991, Mol. Cell. Biol. 11:1785-1792). Retroviral stocks werecharacterized according to their ability to transfer wild type andmutant forms of the BCR-ABL gene product to Rat1 fibroblasts usingimmunohistochemical methods (Muller et al., 1991, Mol. Cell. Biol.11:1785-1792). Endogenous levels of rat c-abl protein were not detectedin these staining procedures. The level of gene transfer was furtherevaluated by measuring levels of BCR-ABL protein expression (Westernblot). Only retroviral stocks showing comparable levels of gene transferwere used in these studies. Titers were in the range of 105 infectiousparticles per ml as determined by the frequency of G418 resistant Rat1colonies following exposure to limiting dilutions of the viral stocks.

6.1.2. Antibodies

Polyclonal rabbit antibodies directed against the amino terminus ofcBCR, and amino- and carboxy-terminal sequences of cABL have beenpreviously described (Pendergast et al., 1991, Cell 66:161-171; Konopkaet al., 1984, Cell 37:1035-1042). A mouse monoclonal anti-ABL (21-63)antibody was employed for immunoblotting (Pendergast et al., 1991, Proc.Natl. Acad. Sci. USA 88:5927-5931). Polyclonal rabbit antibodies to theC-terminal SH3 domain of GRB-2 (Ab50) and to a synthetic peptide derivedfrom the N-terminal SH3 domain of GRB-2 (Ab86) were used forimmunoprecipitation and immunoblotting, respectively (Lowenstein et al.,1992, Cell 70:431-442).

6.1.3. Plasmid Constructions

The 650-base pair human GRB-2 CDNA (Lowenstein et al., 1992, Cell70:431-442; Matuoka et al., 1992, Proc. Natl. Acad. Sci. USA89:9015-9019) was cloned from a human placenta cDNA library bypolymerase chain reaction (PCR) as (5′) SH3-SH2 and (3′) SH3 fragments.A unique KpnI site at codon 154 of GRB-2 was employed to generate thefull length GRB-2 cDNA. The entire coding sequence of GRB-2 and the (3′)SH3 domain of GRB-2 were subcloned in-frame into the Bam HI site of thepGEX-2T vector (Pharmacia). The isolated (5′) SH3 and SH2 domains ofGRB-2 were prepared as described (Skolnik et al., 1993, EMBO J.12:1929-1936).

Preparation of cDNAs for wild type P185 BCR-ABL (McLaughlin et al.,1989, Mol. Cell. Biol. 9:1866-1874) and P185 (Δ176-426) (Muller et al.,1991, Mol. Cell. Biol. 11:1785-1792) and cloning of the correspondingCDNAs into pSRα (Pendergast et al., 1991, Proc. Natl. Acad. Sci. USA88:5927-5931) and pSRα MSVtKneo (Muller et al., 1991, Mol. Cell. Biol.11:1785-1792) vectors was performed as previously described. The P185BCR-ABL (Y177F) mutant was created by oligonucleotide-site directedmutagenesis using the Muta-Gene Phagemid in vitro mutagenesis system(Bio Rad). Template was generated by subcloning a 1.6 Kb EcoRI-SacIfragment of cBCR from pGEM4/cBCR into the EcoRI and SacI sites of thepBlueScript SK+ vector (Stratagene) and rescuing the single stranded DNAby coinfecting XL-1 Blue bacteria with the helper phage R408(Stratagene). The mutagenic oligonucleotide, 5′-AAG CCC TTC TTC GTT AACGTC GAG-3′ (SEQ ID NO: 10), was employed to create a phenylalanine codonin place of tyrosine, by changing nucleotide 530 from an A to a T. Inaddition, a silent base change at nucleotide 534 was introduced tocreate a unique HpaI site. Mutagenized plasmids were selected for thepresence of the unique Hpal site. The mutations were verified by dideoxychain termination sequence analysis in both directions. The mutated BCRsequence was introduced into the wild type P185 BCR-ABL cDNA. P185BCR-ABL (Y177F) was subcloned into the pSRα and pSRα MSVtKneo mammalianexpression vectors and the AcC12 vector for baculovirus expression.Cloning of the cDNA for wild type cABL into the pSRα vector has beenpreviously described (Pendergast et al., Proc. Natl. Acad. Sci. USA88:5927-5931). BCR (Δ872-1271) as also cloned into pSRα as described(Pendergast et al., 1991, Cell 66:161-171).

6.1.4. Expression and Purification of GST-Fusion Proteins

GST-fusion proteins were expressed and purified usingglutathione-Sepharose 4B beads (Pharmacia) as previously described(Pendergast et al., 1991, Cell 66:161-171). Fusion proteins were left onthe resin and stored at 4° C.

6.1.5. Metabolic Labeling and Immunoprecipitation

Sf9 cells were infected with the indicated recombinant baculbviruses.Three days post-infection the cells were incubated with 0.1 mCi/ml [³⁵S]methionine (ICN) in methionine-free media for 4 to 6 hrs at 27° C.Labeled cells were lysed with either KLB (10 mM sodium phosphate pH 7.0,150 mM NaCl, 1% Triton X-100) or PCLB (50 mM HEPES, pH 7.0, 150 mm NaCl,1 mM MgCl₂, 1% Triton X-100, 10% glycerol) supplemented with 5 mM EDTA,1 mM PMSF, 50 μg/ml leupeptin, 25 μg/ml aprotinin, 25 mM NaF, 1 mMNA₃VO₄, and 0.1 mM Na₂MoO₄. The lysates were clarified by centrifugationat 100,000×g for 1 hour. Lysates were incubated with the indicatedantibodies directly or after a 3-fold dilution with Incubation Buffer(20 mM HEPES, pH 7.0, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.5mM Na3VO4, 0.1 mM Na₂MoO₄, 25 mM NaF, ImM PMSF, 25 μg/ml leupeptin) asindicated. Immune complexes were collected by incubation with ProteinA-Sepharose beads (Pharmacia) for 120 minutes at 4° C. The beads werewashed extensively with Incubation buffer to remove unbound material.Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresisand visualized by fluorography. Whenever unlabeled lysates wereemployed, the proteins were detected by immunoblotting with theindicated antibodies.

6.1.6. Binding Assays

Protein lysates were diluted 3-fold with Incubation Buffer and incubatedwith GST or GST-fusion proteins attached to glutathione-Sepharose beads.After incubation for 90 min. at 4° C., the beads were washed extensivelywith Incubation Buffer or with RIPA buffer (20 mM Tris-HCl, pH 7.4, 137mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and2 mM EDTA), as indicated. Bound proteins were analyzed bySDS-polyacrylamide followed by fluorography for radiolabelled proteins.

6.1.7. Immunoblotting, in vitro Autophosphorylation andDephosphorylation Reactions

Procedures for immunoblotting and in vitro labeling with [γ³²P]ATP werecarried out essentially as previously described (Pendergast et al.,1991, Cell 66:161-171; Pendergast et al., 1991, Proc. Natl. Acad. Sci.USA 88:5927-5931). Dephosphorylation with potato acid phosphatase wasperformed as described (Pendergast et al., 1991, Cell 66:161-171;Pendergast et al., 1991, Proc. Natl. Acad. Sci. USA 88:5927-5931).

6.2. Results

6.2.1. BCR-ABL Associates with GRB-2 in vivo

To determine whether the BCR-ABL tyrosine kinase forms a physicalcomplex with the GRB-2 adaptor protein in intact cells, it was examinedwhether the two proteins co-immunoprecipitate. Cell lysates wereprepared from the Ph¹-positive leukemic cell lines K562, MEG-01, andALL-1, and were subjected to immunoprecipitation with anti-ABL,anti-GRB-2, or control antibodies. Following extensive washing, theimmunoprecipitates were incubated in the presence of radioactive ATPunder conditions that promote autophosphorylation of the BCR-ABL kinase.Comparable levels of BCR-ABL protein were precipitated using eitheranti-GRB-2 or anti-ABL antibodies (FIG. 2, lanes 1-9). Both forms ofBCR-ABL, P210 and P185, associated with GRB-2 in Rat1 fibroblastsexpressing the corresponding BCR-ABL proteins (FIG. 2, lanes 10-15).Similarly, immunoprecipitation with anti-GRB-2 can be precipitated byanti-ABL antibodies in cell lines where BCR-ABL is expressed (FIG. 3).Metabolic labelingl of the BCR-ABL expressing cells with ³⁵S-methioninefollowed by immunoprecipitation with anti-ABL or anti-GRB-2 antibodiesdemonstrated that 50% to 90% of the BCR-ABL kinase available in the cellis complexed with GRB-2 in agreement with the results shown in FIG. 2.Interestingly, no association of GRB-2 with the oncogenic v-abl kinase(FIG. 2, lanes 16-18) was observed. These experiments demonstrate thatthe GRB-2 adaptor protein forms a stable complex with both forms of theBCR-ABL tyrosine kinase but not the v-abl kinase and that theBCR-ABL/GRB-2 complexes remain intact following in vitro phosphorylationof BCR-ABL.

6.2.2. BCR-ABL Binds to GRB-2 in vitro

To examine the molecular basis for the association of BCR-ABL withGRB-2, the full-length GRB-2cDNA sequence was cloned into pGEX-2T forexpression in bacteria as a glutathione S-transferase (GST) fusionprotein. The GST-GRB-2 protein was purified and tested for its abilityto bind to BCR-ABL in vitro. It was found that P185 BCR-ABL, expressedin baculovirus-infected insect cells, bound to full length GRB-2immobilized on glutathione-sepharose beads (FIG. 4, lane 2). No bindingto GST alone was detected (FIG. 4, lane 1). The complex of BCR-ABL andGST-GRB-2 remained intact after washing with buffer containing thedetergents SDS and deoxycholate (FIG. 4, lane 2).

To identify which GRB-2 domain(s) bind to BCR-ABL, GST fusion proteinswere prepared that contained the isolated GRB-2 SH3 and SH2 domains.BCR-ABL bound to the GST-GRB-2 SH2 fusion protein (FIG. 4, lane 4). GSTfusion proteins containing the amino- and carboxy-terminal SH3 domainsof GRB-2 also bound to the baculovirus-produced BCR-ABL protein in vitro(FIG. 4, lanes 3 and 5). The interaction of BCR-ABL with the SH2 and SH3domains of GRB-2 was resistant to washing with a buffer containing SDSand deoxycholate (FIG. 4, lanes 3-5). Treatment of BCR-ABL with potatoacid phosphatase completely eliminated its ability to associate with theGRB-2 SH2 domain, but did not affect its binding to the GRB-2 SH3domains.

7. EXAMPLE

BCR-ABL Sequences Necessary for BH2-Mediated Binding of GRB-2 to BCR-ABL

In the Working Example presented in this section, amino acid sequencesnecessary for BCR-ABL/GRB-2 binding via the GRB-2 SH2 domains areinvestigated. Specifically, it is demonstrated that binding requires thepresence of Tyr-phosphorylated amino acid residues, and further, it isshown that a BCR first exon mutation is capable of abolishing theSH2-mediated BCR/GRB-2 binding.

7.1. Materials and Methods

The materials and techniques utilized in the experiments presented inthis Working Example are as those described in Section 6.1, above.

7.2. Results

7.2.1 SH2-Mediated Binding of GRB-2 to BCR-ABL Requires TyrosinePhosphorylation of BCR Sequences

To identify which regions in BCR-ABL participate in GRB-2-binding, BCRand ABL sequences were expressed separately in baculovirus-infectedinsect cells and tested for their ability to bind to full length GRB-2as well as to the isolated GRB-2 SH2 and SH3 domains.Baculovirus-produced cABL bound to full length GRB-2 in vitro (FIG. 5,lane 2) but not to the GRB-2 SH2 domain alone (FIG. 5A, lane 4). cABLhyperexpressed in insect cells is phosphorylated on tyrosine residues(Pendergast et al., 1991, Proc. Natl. Acad. Sci. USA 88:5927-5931).Thus, the inability of the GRB-2 SH2 domain to bind to thebaculovirus-produced cABL protein was not due to lack of tyrosinephosphorylation.

The binding of cABL to GRB-2 in vitro appears to be mediated exclusivelyvia the amino- and carboxy-terminal SH3 domains of GRB-2 (FIG. 5A, lanes3 and 5). SH3 domains bind to specific proline-rich motifs (Cicchetti etal., 1992, Science 257:803-806; Ren et al., 1993, Science259:1157-1161). Recently, it has been shown that GRB-2 binds to the Soslguanine nucleotide exchanger through the direct interaction of the GRB-2SH3 domains with the proline-rich sequence, PPPVPPR, present in thecarboxy-terminal region of Sosl (Li et al., 1993, Nature 363:85-87;Rozakis-Adcock et al., 1993, Nature 363:83-85). The carboxy-terminus ofcABL contains several proline-rich stretches. However, no PPPVPPRsequence is found in the cABL protein. Binding of the GRB-2 SH3 domainsto cABL in vitro is significantly reduced but not completely eliminatedin a cABL deletion mutant protein that lacks the majority of the cABLcarboxy-terminal domain. These data together with the lack of detectableassociation between normal cABL and GRB-2 following immunoprecipitationfrom cell lysates (see FIG. 6A, below), suggest that the cABL/GRB-2interaction observed in vitro is not specific and may not occur in vivo.However, it ismpossible that the proline-rich domain in cABL serves asbinding sites for other SH3 domains.

A similar analysis of the binding of cBCR to full length or isolateddomains of GRB-2 in vitro revealed that only the full length GRB-2 andto a lesser extent the N-terminal SH3 domain of GRB-2 bound to cBCR invitro (FIG. 5B, lanes 2 and 3). Interestingly, no binding of cBCR to theSH2 domain of GRB-2 was detected (FIG. 5B, lane 4).

The complete lack of in vitro binding of the GRB-2 SH2 domain to cABLand CBCR contrasts with the strong binding of this domain to thechimeric BCR-ABL tyrosine kinase (compare FIG. 4 with FIGS. 5A and 5B,lanes 4). The phosphorylation state of cBCR sequences is different inthe full length cBCR protein versus the BCR-ABL chimera. The cBCRprotein is phosphorylated only on serine/threonine residues in all celltypes examined even following hyperexpression in insect cells (Timmonset al., 1989, Oncogene 4:559-567; Pendergast et al., 1991, Cell66:161-171). In contrast, in the BCR-ABL chimera, the activated ABLkinase phosphorylates BCR first exon sequences on tyrosine (Liu et al.,1993, Oncogene 8:101-109). To evaluate whether tyrosine-phosphorylationof BCR sequences could uncover binding to the isolated GRB-2 SH2 domain,insect cells were co-infected with baculoviruses coding for the fulllength cBCR and cABL proteins. Trans-phosphorylation of cBCR by the cABLtyrosine kinase resulted in binding of the GRB-2 SH2 domain to cBCR(FIG. 5C, lane 4). Western blotting, with antiphosphotyrosine antibodiesdemonstrated that CBCR was tyrosine-phosphorylated in Sf9 cellsco-infected with cABL and CBCR baculoviruses. A low level of binding ofcBCR to the isolated amino- and carboxy-terminal SH3 domains of GRB-2 invitro was also detected (FIG. 5C, lanes 3 and 5). These resultsdemonstrate that binding of the GRB-2 SH2 domain to BCR sequencesrequires tyrosine phosphorylation.

7.2.2. GRB-2 Interacts with BCR-ABL but not cABL and CBCR Sequences invivo

To examine whether the in vitro binding of GRB-2 to cABL and cBCR alsooccurred in vivo, COS cells were transfected with expression constructsof the corresponding cDNAs. Transfection of these cDNAs results in anapproximately 50- to 200-fold increase. in the expression of cABL andcBCR over the corresponding endogenous protein levels (Pendergast etal., 1991, Proc. Natl. Acad. Sci. USA 88:5927-5931). Under theseconditions, no appreciable levels of cABL co-immunoprecipitated withGRB-2 (FIG. 6A, lane 6). Similarly, no interaction of GRB-2 with BCRsequences could be detected following hyperexpression of the BCRsequences retained in the P210 BCR-ABL chimera in COS cells (FIG. 6B,lane 3). In contrast, significant levels of P185 BCR-ABL wereprecipitated by the anti-GRB-2 antibodies in the same experiment (FIG.6A, lane 3).

These data, together with the lack of detectable endogenous cABL andCBCR proteins in anti-GRB-2 immunoprecipitates from normal cells,supports the contention that no cABL/GRB-2 and cBCR/GRB-2 complexes maybe found in vivo. These findings demonstrate that the BCR-ABL chimeraexhibits novel protein binding. properties which are distinct from thoseof its BCR and ABL protein sequence components.

7.2.3. A Point Mutation in the BCR First Exon Abolishes Binding of TheGRB-2 SR2 Domain to BCR

Binding of SH2 domains to specific tyrosine-phosphorylated proteins isdependent on the primary sequence C-terminal to the phosphorylatedtyrosine (Fanti et al., 1992, Cell 69:413-423; Kashishian et al., 1992,EMBO J. 11:1373-1382; Sonyang et al., 1993, Cell 72:767-778).Examination of the sequences surrounding the eleven potential tyrosinephosphorylation sites within the first exon of BCR revealed thattyrosine 177 is found within the sequence YVNV, which corresponds to anoptimal binding site for the GRB-2 SH2 domain (Sonyang et al., 1993,Cell 72:767-778; Skolnik et al., 1993, EMBO J. 12:1929-1936). Sequencessurrounding the other ten tyrosines in the BCR first exon do not conformto optimal binding sites for the GRB-2 SH2 domain or other SH2 domainsexamined (Sonyang et al., 1993, Cell 72:767-778). To determine whethertyrosine 177 is required for the binding of BCR-ABL to the GRB-2 SH2domain, phenylalanine was substituted for tyrosine 177 in BCR-ABL bysite-directed mutagenesis, the mutated protein was expressed in insectcells and was tested for binding to GRB-2. Comparison of thephosphopeptide map patterns of the P185 (Y177F) mutant and P185 wildtype proteins following in vitro autophosphorylation revealed theabsence of at least one major phosphopeptide in P185 (Y177F) (FIG. 7).Binding studies demonstrated that in contrast to the wild type P185BCR-ABL, the P185 BCR-ABL (Y177F) mutant protein did not interact withthe GRB-2 SH2 domain in vitro (FIG. 8B, lane 1).

Next, the ability of the P185 BCR-ABL (Y177F) mutant to interact withGRB-2 in vivo was evaluated. P185 (Y177F) did not interact withendogenous GRB-2 when hyperexpressed in COS cells (FIG. 9A, lane 6), orin Rat1 fibroblasts that stably express the mutant protein (FIG. 9B,lane 4). Similarly, BCR-ABL deletion mutants which lack tyrosine 177failed to bind GRB-2. These data indicate that interaction betweenBCR-ABL and GRB-2 in vivo is mediated by the binding of the GRB-2 SH2domain to the phosphorylated tyrosine 177 in BCR-ABL. The GRB-2 SH3domains apparently do not contribute to the in vivo binding between fulllength GRB-2 and BCR-ABL.

8. EXAMPLE

BCR-ABL Proteins Defective in GRB-2 Binding Exhibit DecreasedTransforming Capacity

The data presented in this Working Example demonstrates that thetransformation potential of the BCR-ABL intracellular PTK is dependentupon the binding of this PTK to the GRB-2 member of the GRB subfamily ofthe adaptor family of proteins. This result represents the first case inwhich the binding of an adaptor protein to such a PTK is implicated as astep in a transformation/oncogenesis process.

8.1. Materials and Methods

8.1.1. Transformation Assays

Infection of Rat1 fibroblasts was carried out as previously described(Lugo et al., 1989, Mol. Cell. Biol. 9:1263-1270). Titers were in therange of >10⁵ infectious particles per ml as determined by the frequencyof gene transfer to fibroblasts. Rat1 colony formation in semisolidmedium was measured by plating 5×10⁴ cells per 6-cm² dish in 5 ml ofIscoves supplemented with 15% fetal calf serum and 0.4% Noble agar (Lugoet al., 1989, Mol. Cell. Biol. 9:1263-1270). G418-resistant populationswere established by culturing the infected cells for 12-15 days in G518(0.5 mg per ml). Following selection, cells from G418-resistant cultureswere plated in agar at a density of 104 cells per ml. The number ofcolonies formed in the agar was recorded two weeks after plating thecells.

Infection and establishment of hematopoietic cell cultures from freshlyisolated murine bone marrow were performed as previously described(McLaughlin et al., 1987, Proc. Natl. Acad. Sci. USA 84:6558-6562). Inbrief, bone marrow elements from 4-6 week old female BALB/c mice wereresuspended at 2×10⁶ cells per ml in the appropriate retroviral stocksupplemented with 4 μg of Polybrene per ml. Cells were incubated for 4hours at 37° C. Following infection, cells were washed and resuspendedin RPMI 1640 medium supplemented with 5% fetal calf serum and 50 μMβ-mercaptoethanol, at a density of 10⁶ cells per ml 5 ml of the cellsuspension was plated into each 6 cm² dish. Cultures were maintained forup to 8 weeks and fresh media was added once a week. The cultures wereconsidered transformed when the cell density of the nonadherenthematopoietic cells exceeded 10⁶ cells per ml.

8.1.2. Miscellaneous Procedures

Techniques in addition to the transformation assays as described inSection 8.1.1 are as those described, above, in Section 6.1.

8.2. Results

To determine the biological relevance of GRB-2 binding toBCR-ABL-induced oncogenesis, the effect of mutating tyrosine 177 wasexamined, required for association with GRB-2, on the ability of BCR-ABLto transform fibroblasts and hematopoietic cells. Helper-free retroviralstocks of wild type and mutant BCR-ABL forms were used to infect Rat1fibroblasts and freshly isolated murine bone marrow cells. Both GRB-2and hSos-I have been shown to be expressed in these cell types(Lowenstein et al., 1992, Cell 70:431-442; Bowtell et al., 1992, Proc.Natl. Acad. Sci. USA 89:6511-6515; Chardin et al., 1993, Science260:1338-1343). Transformation of Rat1 fibroblasts was assayed by colonyformation in soft agar (Lugo et al., 1989, Mol. Cell. Biol.9:1263-1270). Hematopoietic cell transformation was assayed by culturinginfected mouse bone marrow cells under conditions that support thegrowth of pre-B lymphocytes (McLaughlin et al., 1987, Proc. Natl. Acad.Sci. USA 84:6558-6562). In contrast to wild type BCR-ABL, the P185(Y177F) protein did not transform hematopoietic cells and exhibited adecreased capacity to transform Rat1 cells (FIG. 10). These results areconsistent with the previous observation that the P185 (Δ176-426) mutantwhich deletes Y177 exhibited decreased transforming activity in bothRat1 fibroblasts and hematopoietic cells (Muller et al., 1991, Mol.Cell. Biol. 11:1785-1792; FIG. 10). P185(Δ176-426) displays a morepronounced deficiency in the transformation of Rat1 fibroblasts thanP185 (Y177F), particularly after G418 selection (FIG. 10). Sequencesdownstream of Y177 in the BCR first exon have been shown to bind to SH2domains in a phosphoserine/phosphothreonine-dependent manner (Pendergastet al., 1991, Cell 66:161-171; Muller et al., 1991, Mol. Cell. Biol.11:1785-1792). Two SH2-binding sites have been identified within thisregion (designated A and B) and removal of these sites may abolishspecific protein interactions important for BCR-ABL-mediatedtransformation of fibroblasts. Both P185 (Y177F) and P185 (Δ176-426)showed no transformation activity in hematopoietic cells. Differences inthe transforming activities of the BCR-ABL proteins were not due todifferent levels of protein expression. Western blot analysis of lysatesfrom cells expressing the various BCR-ABL forms revealed comparablesteady-state levels of the proteins following infection of Rat1fibroblasts with the corresponding retroviruses (FIG. 11).

9. EXAMPLE

BCR-ABL Activities Transcription from a RAS-Responsive Element Promoterin a RAS-Dependent Manner

In the Working Example presented in this section, it is demonstratedthat BCR-ABL intracellular PTK/GRB-2 binding serves to activate the Rassignalling pathway.

9.1. Materials and Methods

9.1.1. Transcriptional Activation Assay

Transcriptional activation of expression from a Ras-responsive element(ets/AP-1) promoter was done essentially as described previously (Clarket al., 1993, Proc. Natl. Acad. Sci. USA 90:4887-4891; Hauser et al.,1993, Methods in Enzymology, in press). Briefly, NIH 3T3 cells weretransfected with 1 μg of the pB4X-CAT chloramphenicol acetyl transferasereporter plasmid (Wasylyk et al., 1989, Mol. Cell. Biol. 9:2247-2250),together with 0.5 μg pSRαMSVTkneo containing the indicated BCR-ABLmutants in the presence or absence of 5 μg pZIP H-Ras (17N) (Feig etal., 1988, Mol. Cell. Biol. 8:3235-3243). Transfections were performedin duplicate in 60 mm dishes and the cells harvested after 48 hr.Following lysis by freeze thaw in 100 μl of 250 mM Tris HCl (pH 7.8),cell debris was removed by centrifugation and the supernatant heated to62° C. to denature endogenous acyl transferases. Following furthercentrifugation, a 50 μl aliquot of each supernatant was assayed for CATactivity by incubation with 0.1 μCi of ¹⁴C chloramphenicol (NEN) and0.34 mM acetyl CoA i 250 mM Tris-HCl in a final reaction volume of 140μl for 45 minutes. The reaction was then terminated by extraction with500 μl of ethyl acetate, evaporated under vacuum, and the resultingpellets were redissolved and subjected to thin layer chromatography onsilica gel plates, using 5% methanol/95% chloroform (v/v) as solvent.Assays were quantitated using an AMBIS beta scanner.

9.1.2. Miscellaneous Techniques

Techniques in addition to the transformation assays as described inSection 8.1.1 are as those described, above, in Section 6.1.

9.2. Results

A mechanism whereby GRB-2-binding to BCR-ABL may potentiate oncogenictransformation is through direct stimulation of Ras via the Sos-1guanine nucleotide exchange factor. To examine whether the interactionof BCR-ABL with GRB-2 feeds directly into the Ras pathway atranscriptional activation assay has been employed. Oncogenic Rasincreases the rate of transcription from Ras-responsive elements (e.g.ets-1 and AP-1 DNA motifs; Hauser et al., 1993, Methods in Enzymology,in press). In addition, oncogenes with a wide range of functions,including protein tyrosine kinases and serine/threonine kinases canactivate transcription from promoters containing Ras-responsive elementssuch as the ets/AP-1 motif (Schweighoffer et al., 1992, Science256:825-827). A correlation exists between the ability of variousoncogenes to activate transcription from an ets/AP-1-containing promoterand their capacity to transform cells.(Wasylyk et al., 1988, EMBO J.7:2475-2483). Thus, transactivation assays may complement the cellgrowth and tumorigenicity studies for the analysis of oncogene function.

Wild type and mutant forms of BCR-ABL were compared for their ability toactivate transcription from ets/AP-1. A CAT reporter under the controlof a β-globin promoter that contains four tandem Ras-responsive elements(pB4X-CAT). Oncogenic Ras has been shown to increase the rate oftranscription from this element up to 15-fold (Schweighoffer, Science256:825-827). As shown in FIG. 12, wild type BCR-ABL-induced activationis abolished by co-transfection of Ras (17N), a dominant inhibitorymutant which has been shown to neutralize Ras function (Feig et al.,1988, Mol. Cell. Biol. 8:3235-3243; Thomas et al., 1992, Cell68:1031-1040; Wood et al., 1992, Cell 68:1041-1050) indicating thatBCR-ABL-induced transcriptional activation is mediated by Ras.Significantly, the BCR-ABL mutants, P185 (Y177F) and P185 (Δ176-426),that are deficient for GRB-2 binding, produced little, if any, effect ontranscriptional transactivation from this promoter (FIG. 11). The lowlevel of transactivation obtained with the BCR-ABL mutants correlateswith their decreased transforming activities compared to wild type P185BCR-ABL (FIG. 10). Further, infection of Rat1 fibroblast cells with wildtype BCR-ABL-containing retrovirus produces an increase in the fractionof Ras-GTP (FIG. 13). Quantitation of the amount of GDP and GTP bound toRas with an AMBIS beta scanner shows a modest but reproducible increase.

10. EXAMPLE

Signaling Incompetent GRB2 Reverses the Phenotype of Transformed Cells

The following example shows that disruption of the signal transductionpathway involving BCR/ABL and GRB2 can reverse a transformed phenotypein cells. Elevated expression of signaling incompetent GRB2 mutantsreverses BCR/ABL induced transformed growth in Rat 1 cells. Disruptionof the signaling pathway also inhibits the growth of cells isolated froma patient with chronic myelogenous leukemia (K562 cells) that expressBCR/ABL as well as transformed cells dependant on the PDGF receptor.

10.1. Materials and Methods

Mutant GRB2 genes were constructed that deleted the SH3 domain on eitherthe amino (N′) or carboxy (C′) terminus. Full length and truncated formsof GRB2 were inserted into the Bam H1 site of a modified pCGN plasmidvector (Tanaka et al., Cell 60:375-386, 1990) as described in Pendergastet al., Cell 75:175-185, 1993. This vector has the hygromycin resistancegene inserted upstream of the SV40 origin of replication. To facilitatediscrimination of the endogenous GRB2 protein from the transfectedconstructs, a sequence coding for the influenza virus hemagglutininepitope (Pati, U. K., Gene 114:285-288, 1992) was fused in frame at theN terminus of the mutant proteins. The same antigenic tag was fused tothe wild type GRB2 gene.

BCR/ABL transformed Rat 1 fibroblasts were established as described inExample 8.1.1 above. GRB2 constructs were transfected into Rat1-BCR/ABLcell lines and the rat glioman cell line C6 (ATCC CCL 107) using thestandard calcium phosphate transfection protocol (Molecular CloningTechniques, Laboratory Manual, 2nd Ed., Ed Sambrook et al., Cold-SpringHarbor Laboratory Press, 1989 and Muller). Following transfection, theRat1 cells were cultured for 24 hrs, then exposed to the drugsG418+hygromycin (Sigma) for 7-9 days. The double drug selected masspopulation of cells were seeded in soft agar and the levels ofendogenous BCR/ABL, GRB2 and transfected GRB2 determined using Westernblot. Individual clonal cell lines expressing the different transfectedGRB2 constructs were also established. In the case of transfected C6cells, cells were selected in hygromycin containing media for 10 days asdescribed in Muller, supra. Following drug selection, cells were seededin soft agar medium containing 0.5% FCS and PDGF (1 ng/ml).Colonies >0.4 mm were counted on day 10.

Expression of the GRB2 constructs was evaluated by immunoprecipitationas described in Pendergast et al., Cell 75:175-185, 1993. Briefly,BCR/ABL-GRB2 expressing Rat1 cells were lysed and immunoprecitipatedwith anti-ABL pEx4 antibodies and the immunoprecipitates were subjectedto an in vitro autokinase assay (Pendergast et al., supra). The sampleswere analyzed by SDS-PAGE gel and visualized by autoradiography. In asecond experiment, cells were lysed in 10 mM Tris-HCl, ph 7.4, 1% SDS, 1mM PMSF, 15 μg of protein from each sample was separated using SDS-PAGE(15%). The proteins were electrophoretically transferred tonitrocellulose filters and immunoblotted with an anti-GRB2 mousemonoclonal antibody (Transduction Laboratories) followed by incubationwith goat anti mouse Mab conjugated to horse-radish peroxidase (BioRad).Proteins were visualized with the Enhanced Chemiluminescence detectionsystem (Amersham).

Soft agar assays were performed as previously described (Lugo et al.,Mol. Cell. Bio. 9:1263-1270, 1989). Mass populations of drug selectedcells were seeded at densities ranging between 10³-5×10⁴ cells/6 cm²dish depending on the cloning efficiency of each cell type. Samples wereplated in duplicate in medium containing 20% fetal calf serum.Macroscopic colonies (>0.4 mm) were counted after 14 days. The datarepresents the average number of colonies observed in 3 to 5 independentexperiments performed with mass populations of transfected and drugselected cells. Data for the growth of Rat1-BCR/ABL was derived usingthree independent Rat1-BCR/ABL clonal cell lines transfected with theappropriate constructs and repeated in 2 to 3 separate experiments foreach cell line. Thus the data represents the average number of coloniesobserved in 7 independent experiments.

K562 (ATCC CRL 243) cells were transfected with the GRB2 constructsusing standard methods of electroporation. Following transfection, cellswere cultured for 18 to 24 hrs, then exposed to hygromycin (500 μg/ml)for 2 days. After drug selection numbers of viable cells were determinedby counting cells which exclude the dye trypan blue. Equal numbers ofviable cells were seeded in soft agar.

10.2 Results 10.2.1. Expression of GRB2 in RAT 1 Cells

Expression of the N′GRB2 mutant protein in Rat1-BCR/ABL cells causes areversion to a normal phenotype. The mass population of N′ GRB2 mutantexpressing cells grew as a monolayer in liquid culture and exhibitedcontact inhibition when reaching confluence. Significantly, N′GRB2mutant expressing Rat1-BCR/ABL cells exhibited a dramatic reduction intheir ability to form colonies in soft agar when compared to cellsexpressing the empty vector (see Table 1). Cells expressing the C′GRB2mutant expressing cells grew as monolayers in liquid culture but did notquiesce when confluent. Growth of C′GRB2 mutant expressing Rat1-BCR/ABLfibroblasts in soft agar was also suppressed, albeit to a lesser extentthan that observed for the N′ GRB2 mutant. Because comparable ratios ofmutant proteins to wild type GRB2 protein were present in both the N′and C′ expressing cell population, the greater potency of the N′ mutantto suppress BCR/ABL induced transformation is probably not simply due todifferences in the level of the two mutants (FIG. 14B). Rather thesedifferences may indicate that the two SH3 domains bind SOS withdifferent affinities or bind to different substrates. Thus GRB2 SH3deletion mutant proteins act as dominant negative inhibitors of BCR/ABLinduced transformation when present in equimolar or greater amounts thanthe endogenous wild type GRB2 in the cell.

In contrast to the N′ or C′ GRB2 mutants, elevated expression of wildtype GRB2 in Rat1-BCR/ABL fibroblasts appeared to accentuate thetransformed phenotype. The drug selected mass population of cellsexpressing the transfected wild type GRB2 construct consistentlyexhibited a 25-30% increase in the number of colonies in soft agar whencompared to cells transfected with the empty vector (Table 1). Thesedata suggest that the GRB2 protein may be a limiting effector in BCR/ABLinduced transformation of Rat 1 fibroblasts.

TABLE 1 Construct Rat1 K562 C6 empty vector 566 187 520 wild type GRB2734 209 577 N′ truncated GRB2 14 13 2 C′ truncated GRB2 119 86 3 (#Colonies/1 × 10⁴ cells seeded)

10.2.2. Expression of GRB2 Mutants in K562 Cells

The SH3 GRB2 mutants also inhibit the growth of human leukemic cells.The GRB2 constructs were introduced into K562 cells, a p210 BCR/ABLexpressing cell line established from a CML patient in blast crisis.Following transfection and 48 hrs of drug selection, levels ofendogenous BCR/ABL, GRB2 and transfected GRB2 were determined by Westernblot (FIG. 16). At the same time, viable cells were seeded in soft agar.Cultures seeded with K562 cells expressing the N′GRB2 mutant proteinshowed 10 times fewer the number of soft agar colonies when compared tomock transfected controls (Table 1). Analogous to what was observed inthe Rat1-BCR/ABL fibroblasts, the C′GRB2 mutant also inhibited K562colony formation, but to a lesser degree than the N′ mutant. These dataindicate that signal transduction mediated by GRB2 is an essentialcomponent in BCR/ABL induced human malignancies.

10.2.3. Expression of GRB2 in C6 Gliona cells

The GRB2 constructs were also introduced into rat C6 glioma cells, whichare dependant on PDGF receptor activity for their transformed growthphenotype (Zhang et al., Neurol. Res. 14(5):397-401, 1992). PDGFreceptor has been shown to interact with GRB2 (Lowenstein et al., 1992,Cell 70:431-442). As shown in Table 1, expression of the signalingincompetent GRB2 mutants significantly inhibited colony formation. Thisdata demonstrates that inhibition of GRB2 signal transduction is can beused to inhibit transformed cell growth that is dependant on receptortyrosine kinase activation.

11. EXAMPLE

Signaling Incompetent GRB2 Prevents Tumor Growth in an Animal Model

This example demonstrates that cells expressing signaling incompetentGRB2 are no longer able to form tumors in animals, suggesting thatdisruption of the, BCR/ABL signaling pathway can be used to treat cancerin mammals.

11.1. Materials and Methods

GPB2 (wild type and mutants) and BCR/ABL expressing Rat 1 cells wereprepared as described in Example 10. Nude mice (4 mice per group) wereinjected subcutaneously in the left flank with Rat 1 cells co-expressingp210 BCR/ABL and either wild type GRB2, N′ truncated CRB2 or C′truncated GRB2. Cells expressing p210 BCR(ABL alone (2×10⁶ cells in 100μl) were injected subcutaneously on the right hind leg of each mouse toact as an internal control. The mice were sacrificed at 3 weekspost-implant and the tumor volume measured.

11.2. Results

The results shown in Table 2 clearly demonstrate that expression ofsignaling incompetent GRB2 proteins reduces growth of tumor cells invivo.

TABLE 2 Group left right wild tpe GRB2 0.5 0.5 N′ truncated 0.3 0.7 GRB2C′ truncated <0.1 0.7 GRB2 (data shown is average tumor size per group)

12. EXAMPLE

Effects of Signaling Incompetent GRB2 on RAS Activation

This example shows that the signaling incompetent GRB2 moleculesdescribed herein inhibit BCR/ABL induced ras activation in proportion totheir transformation inhibitory potency suggesting that interactionbetween GRB2 and downstream signaling components that lead to rasactivation is disrupted.

12.1. Materials and Methods

Transcriptional activation of expression from a Ras-responsive element(ets/AP-1) promoter was done as described in Example 9. Transformationof Rat1 cells, soft agar assays and immunoblotting experiments wereperformed as described in Example 10.

12.2. Results

To determine whether the growth inhibitory effect of the GRB2 mutantswas due to their ability to block ras activation, lysates prepared fromRat1-BCR/ABL cell lines hyperexpressing wild type or mutant GRB2proteins were evaluated for their ability to activate ras. Using a rasdependent transcription activation assay, we observed that lysates fromN′ GRB2 expressing Rat1-BCR/ABL cells exhibited a 10-15 fold lower rasactivation activity than mock transfected controls (FIG. 15). Lysatesfrom C′ GRB2 expressing cell lines also had a decrease in their rasactivation activity, albeit to a lesser extent than that of the N′mutant. The difference in the inhibitory effect of the N′ and C′ mutantsis consistent with their potency to reverse the BCR/ABL transformedphenotype.

If the GRB2 mutant proteins function by blocking intermediate steps inthe signal transduction pathway prior to ras, then transformation bygenes which circumvent ras activation should not be effected. To testthis hypothesis, the GRB2 constructs were transfected into Rat 1 cellstransformed with an activated form of raf, a serine kinase which elicitsits mitogenic effect downstream of ras in the same signaling pathway(Kolch et al., Nature 349:426, 1991). Following drug selection withhygromycin the mass population of cells were seeded in agar. Rat1/v-rafcells expressing N′ or C′ GRB2 mutants gave rise to the same number ofcolonies in soft agar as the mock transfected control cells (Table 3).Furthermore, cell lines expressing a greater than two fold excess of SH3mutant than endogenous wild type GRB2 protein were not inhibited intheir ability to grow in soft agar (FIG. 16). These data suggest thatthe GRB2 mutant proteins inhibit the BCR/ABL mitogenic signal byuncoupling the signal transduction pathway upstream of raf.

TABLE 3 Construct Rat1/v-raf empty vector 620 wild type GRB2 665 N′truncated GRB2 653 C′ truncated GRB2 598 (# Colonies/1 × 10⁴ cellsseeded)

It is apparent that many modifications and variations of this inventionas set forth here may be made without departing from the spirit andscope thereof. The specific embodiments described hereinabove are givenby way of example only and the invention is limited only by the terms ofthe appended claims.

17 1 6 PRT Artificial Sequence Description of Artificial Sequenceconsensus sequence 1 Asp Leu Arg Ala Ala Asn 1 5 2 6 PRT ArtificialSequence Description of Artificial Sequence consensus sequence 2 Asp LeuAla Ala Arg Asn 1 5 3 8 PRT Artificial Sequence Description ofArtificial Sequence consensus sequence 3 Pro Xaa Xaa Trp Xaa Ala Pro Glu1 5 4 6 PRT Artificial Sequence Description of Artificial Sequenceconsensus sequence 4 Phe Leu Ile Arg Glu Ser 1 5 5 6 PRT ArtificialSequence Description of Artificial Sequence consensus sequence 5 Phe LeuVal Arg Glu Ser 1 5 6 7 PRT Artificial Sequence Description ofArtificial Sequence consensus sequence 6 Xaa Pro Xaa Tyr Xaa Asn Xaa 1 57 10 PRT Artificial Sequence Description of Artificial Sequenceconsensus sequence 7 Xaa Pro Xaa Xaa Pro Pro Pro Xaa Xaa Pro 1 5 10 8 8PRT Artificial Sequence Description of Artificial Sequence consensussequence 8 Pro Pro Pro Val Pro Pro Arg Arg 1 5 9 11 PRT ArtificialSequence Description of Artificial Sequence consensus sequence 9 Xaa HisCys Xaa Ala Gly Xaa Xaa Arg Xaa Gly 1 5 10 10 24 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 10 aagcccttcttcgttaacgt cgag 24 11 4 PRT Artificial Sequence Description ofArtificial Sequence conserved motif 11 Val Ala Val Lys 1 12 5 PRTArtificial Sequence SITE (3) Xaa=any amino acid residue 12 Gly Met XaaTyr Leu 1 5 13 9 PRT Artificial Sequence Description of ArtificialSequence conserved motif 13 Ile His Arg Asp Leu Ala Ala Arg Asn 1 5 14 6PRT Artificial Sequence Description of Artificial Sequence conservedmotif 14 Lys Trp Met Ala Pro Glu 1 5 15 6 PRT Artificial SequenceDescription of Artificial Sequence conserved motif 15 Lys Trp Thr AlaPro Glu 1 5 16 6 PRT Artificial Sequence Description of ArtificialSequence conserved motif 16 Phe Trp Tyr Ala Pro Glu 1 5 17 7 PRTArtificial Sequence Description of Artificial Sequence conserved motif17 Ser Asp Val Trp Ser Phe Gly 1 5

What is claimed is:
 1. A method for identifying a compound that disruptsa protein tyrosine phosphatase polypeptide/GRB-2 adaptor polypeptidecomplex comprising: (a) contacting a cell that forms the complex with acompound that acts intracellularly at a concentration sufficient todisrupt the protein tyrosine phosphatase polypeptide/GRB-2 complex; (b)detecting the level of the complex present in the cell of step (a); and(c) comparing the level of the complex detected in step (b) to the levelof complex present in a cell of the type in step (a) that has notcontacted the compound, so that if the level detected in step (b) isless than the level present in the cell, the compound that disrupts aprotein tyrosine phosphatase polypeptide/GRB-2 adaptor polypeptidecomplex is identified.
 2. The method of claim 1, wherein the GRB-2adaptor polypeptide comprises a F-L-I-R-E-S (SEQ ID NO:4) or F-L-V-R-E-S(SEQ ID NO:5) amino acid sequence.
 3. The method of claim 1, wherein theprotein tyrosine phosphatase polypeptide comprises a X-P-X-Y-V/I-N-V/I(SEQ ID NO:6) amino acid sequence.
 4. The method of claim 1, whereinsaid protein tyrosine phosphatase polypeptide is selected from the groupconsisting of CD45, LAR, RPTPα, RPTPβ, RPTPχ, and RPTPκ.
 5. A method foridentifying a compound to be tested for an ability to disrupt a proteintyrosine phosphatase polypeptide/GRB-2 adaptor polypeptide complexcomprising: contacting a protein tyrosine phosphatase polypeptide or aGRB-2 adaptor polypeptide with a test compound for a time sufficient toallow binding of the test compound to the protein tyrosine phosphatasepolypeptide or the GRB-2 adaptor polypeptide; so that if the testcompound binds the protein tyrosine phosphatase polypeptide or the GRB-2polypeptide, a compound to be tested for an ability to disrupt a proteintyrosine phosphatase/GRB-2 adaptor polypeptide complex is identified. 6.The method of claim 5, wherein the GRB-2 adaptor polypeptide comprises aF-L-I-R-E-S (SEQ ID NO:4) or F-L-V-R-E-S (SEQ ID NO:5) amino acidsequence.
 7. The method of claim 5, wherein the protein tyrosinephosphatase polypeptide comprises X-P-X-Y-V/I-N-V/I (SEQ ID NO:6) aminoacid sequence.
 8. The method of claim 5, wherein said protein tyrosinephosphatase polypeptide is selected from the group consisting of CD45,LAR, RPTPα, RPTPβ, RPTPχ, and RPTPκ.